Expression of trehalose 6-phosphate synthase and trehalose 6-phosphate phosphatase in plant[[s]] plastids

ABSTRACT

The invention provides novel transgenic plants which express trehalose biosynthetic genes, e.g., under control of an inducible promoter, which are developmentally normal, together with methods for improving stress tolerance in said plants, methods of improving food quality, and other methods of making and using the plants of the invention. The invention also provides nucleotide sequences encoded novel trehalose biosynthetic enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/077,665, filed Mar. 11, 1998. The above applicationis incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to expression of trehalose biosyntheticgenes and drought resistance in plants.

BACKGROUND OF THE INVENTION

[0003] Trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside) is adisaccharide commonly found in lower organisms such as bacteria, fungiand insects where it often accumulates in resting or stationary phasecells and organs. Two enzymatic activities are required for trehalosebiosynthesis: a trehalose-6-phosphate synthase catalyses thecondensation of UDP-glucose and glucose-6-phosphate totrehalose-6-phosphate and a trehalose-6-phosphate phosphatasephosphorylates trehalose-6-phosphate to trehalose.

[0004] Although trehalose can serve as a storage form for reducedcarbon, it may play a more significant role as a protectant against thedeleterious effects of various abiotic stresses, notably heat anddesiccation. Both in vivo and in vitro, trehalose accumulation iscorrelated with protection of biological macromolecules (particularlymembranes and proteins) from dessication, temperature extremes, andosmotic shock. Trehalose produced by fermentation is used commerciallyin the preservation of enzymes and is registered as a food additive forthe stabilization of dehydrated and processed foods.

[0005] While it has long been recognized that trehalose may occur inplants as a product of symbiotic microorganisms, as a rule vertebratesand higher plants were thought not to be capable of synthesizingtrehalose. The near-ubiquitous occurrence of specifictrehalose-catabolizing enzymes (trehalases) in higher plant families wasa biological curiousity ascribed mainly to the presence of exogenoustrehalose entering plant cells from symbiotic or epiphytic microbial andfungal sources. Notable exceptions are the lower plants and angiospermsgrouped loosely into the category of “resurrection plants” which arecapable of surviving extraordinarily prolonged periods of dessication.These plants, including species of Selaginella and Myrothamnus, canaccumulate as much as 10% trehalose by dry weight following the onset ofdroughting.

[0006] In view of trehalose's association with drought resistance andthe historically poor economics of microbial trehalose fermentation,attempts have also been made to engineer transgenic plants to accumulatethis disaccharide. Although such plants have been obtained, it hasbecome apparent that constitutive trehalose production in the plantcytosol is accompanied by significant deleterious effects. Thesephenotypes (stunted growth, abnormal leaves, undeveloped roots) areparticulary severe when trehalose expression occurs in root tissue orduring early development, as the use of green-tissue specific plantpromoters to drive trehalose producing genes ameliorates some, but notall, of these effects.

[0007] Given these facts, an inducible expression system for thetrehalose biosynthetic genes, which allows for trehalose accumulationand results in drought resistance but without deleterious effects to theplant is of great practical use and economic interest.

SUMMARY OF THE INVENTION

[0008] The present invention thus relates to expression of trehalosebiosynthetic genes and drought resistance in plants. In particular, thisinvention addresses the issue of trehalose accumulation and droughtresistance in higher plants and novel ways to engineer such trait. Italso addresses the need for improved storage properties of harvestedplants, improved shelf-life of fruits and flowers, as well asstabilization of foreign proteins expressed in transgenic plants. In aprefered embodiment, the invention describes the expression of thetrehalose biosynthetic genes in plants, preferably under the control ofan inducible promoter, which allow for drought resistance without thedeleterious effect associated with uncontrolled accumulation oftrehalose. A prefered promoter is a chemically inducible promoter, suchas the tobacco PR-1a promoter, which can be activated by foliarapplication of a chemical inducer.

[0009] Additionally, the invention describes expression of the trehalosebiosynthetic genes expressed in different cellular compartments. In afirst embodiment the trehalose biosynthetic genes are expressed in theplant cytoplasm. In a further embodiment, the trehalose biosyntheticgenes are expressed from the plant nuclear genome and the trehalosebiosynthetic enzymes encoded therefrom are targeted to the plastids,e.g. by using a plastid transit peptide. In a further embodiment, thetrehalose biosynthetic genes are expressed from the plant plastidgenome. In a preferred embodiment, vectors containing the trehalosebiosynthetic genes fused to a promoter capable of directing theexpression of the trehalose biosynthetic genes in plant plastids aretransformed into the plastid genome. In a preferred embodiment, vectorscontaining a phage promoter fused to the trehalose biosynthetic genesare transformed into the plastid genome. The resulting line is crossedto a transgenic line containing a nuclear coding region for a phage RNApolymerase supplemented with a plastid-targeting sequence and operablylinked to a plant promoter, such as an inducible promoter, atissue-specific promoter or a constitutive promoter. In anotherpreferred embodiment, a promoter capable of directing the expression ofthe trehalose biosynthetic genes in plant plastids is a promotertranscribed by a RNA polymerase normally present in plastids, such as anuclear-encoded polymerase or a plastidencoded polymerase. Suchpromoters are e.g. but not limited to a clpP promoter, a 16S r-RNA genepromoter, a psbA promoter or a rbcL promoter.

[0010] In the present invention, trehalose biosynthetic genes from E.coli are preferably used, but trehalose biosynthetic genes from otherorganisms including but not limited to yeast, other lower organisms orhigher organisms, such as plants, are suitable. In a preferredembodiment, a nucleotide sequence encoding a trehalose phosphatesynthase and a nucleotide sequence encoding a trehalose phosphatephosphatase are both expressed in the plant. In another preferredembodiment, a nucleotide sequence encoding a trehalose phosphatesynthase is expressed in the plant, or a nucleotide sequence encoding atrehalose phosphate phosphatase is expressed in the plant. The presentinvention also relates to the expression from the plastid genome of twotrehalose biosynthetic genes transcribed from a single promoter in anoperon-like polycistronic gene.

[0011] The invention thus provides:

[0012] A plant expressing a nucleotide sequence encoding a trehalosebiosynthetic enzyme, for example a plant comprising nucleotide sequencecoding for the trehalose 6-phosphate synthase and/or trehalose6-phosphate phosphatase, for example the E. coli OtsA and/or E.coli OtsBgenes. Such nucleotide sequences are for example stably integrated inits nuclear or plastid genome, under the control of a promoter capableof directing the expression of the trehalose biosynthetic genes in saidplant, e.g. under the control of an inducible promoter, e.g., awound-inducible or chemically inducible promoter, such as the tobaccoPR-1a promoter or Arabidopsis PR-1 promoter, or, atransactivator-regulated promoter wherein the correspondingtransactivator is under the control of a promoter capable of directingthe expression of the transactivator in said plant, e.g. an induciblepromoter, a tissue-specific promoter or a constitutive promoter, e.g., awound-inducible or chemically inducible promoter, such as the tobaccoPR-1a promoter or Arabidopsis PR-1 promoter;

[0013] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag with instructions for use. In particular, the inventionprovides:

[0014] A plant comprising in its nuclear genome a first heterologousexpression cassette or parts thereof comprising a nucleotide sequenceencoding a trehalose 6-phosphate synthase under control of an induciblepromoter and a second heterologous expression cassette or parts thereofcomprising a nucleotide sequence encoding a trehalose-6-phosphatephosphatase under control of an inducible promoter. also including theseed for such a plant, which seed is optionally treated (e.g., primed orcoated) and/or packaged, e.g. placed in a bag with instructions for use.

[0015] The invention further provides:

[0016] A plant comprising in its plastid genome a first heterologousexpression cassette or parts thereof comprising a nucleotide sequenceencoding a trehalose 6-phosphate synthase under control of a promotercapable of directing the expression of the nucleotide sequence in theplastids of said plant, e.g. a transactivator-regulated promoter whereinthe corresponding transactivator is preferably under the control of aninducible promoter, a tissue-specific promoter or a constitutivepromoter, and a second heterologous expression cassette or parts thereofcomprising a nucleotide sequence encoding a trehalose-6-phosphatephosphatase under control of a promoter capable of directing theexpression of the nucleotide sequence in the plastids of said plant,e.g. a transactivator-regulated promoter wherein the correspondingtransactivator is preferably under the control of an inducible promoter,a tissue-specific promoter or a constitutive promoter.

[0017] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag with instructions for use.

[0018] The invention further provides:

[0019] A heterologous plant nuclear expression cassette comprising anucleotide sequence encoding a trehalose-6-phosphate synthase,preferably under the control of an inducible promoter, e.g., a woundinducible or chemically inducible promoter;

[0020] a vector comprising such plant expressible cassette; and

[0021] a plant transformed with such a vector.

[0022] The invention further provides:

[0023] A heterologous plant nuclear expression cassette comprising anucleotide sequence encoding a trehalose-6-phosphate phosphatase,preferably under the control of an inducible promoter, e.g., a woundinducible or chemically inducible promoter;

[0024] a vector comprising such plant expressible cassette; and

[0025] a plant transformed with such a vector.

[0026] The invention further provides:

[0027] A heterologous plant expression cassette comprising a nucleotidesequence encoding a trehalose-6-phosphate synthase, preferably under thecontrol of an inducible promoter, e.g., a wound inducible or chemicallyinducible promoter, and further comprising a nucleotide sequenceencoding a trehalose-6-phosphate phosphatase, preferably under thecontrol of an inducible promoter, e.g., a wound inducible or chemicallyinducible promoter;

[0028] a vector comprising such plant expressible cassette; and

[0029] a plant transformed with such a vector.

[0030] In a further embodiment, the invention encompasses expression ofnucleotide sequences encoding trehalose biosynthetic enzymes in plastidsunder the control of a promoter capable of directing the expression of anucleotide sequence in the plastids of a plant, e.g. atransactivatorregulated promoter, and the gene for the transactivator isin the nuclear DNA, under the control of an plant promoter. For example,plastid transformation vectors are typically constructed using a phagepromoter, such as the phage T7 gene 10 promoter, the transcriptionalactivation of which is dependent upon an RNA polymerase such as thephage T7 RNA polymerase. The resulting line is crossed to a transgenicline containing a nuclear coding region for a phage RNA polymerasesupplemented with a chloroplast-targeting sequence and operably linkedto a plant promoter, preferably an inducible promoter, a tissue-specificpromoter or a constitutive promoter, preferably a chemically induciblepromoter such as the tobacco PR-1a promoter.

[0031] The invention thus additionally provides:

[0032] A plant comprising

[0033] a heterologous plastid expression cassette or parts thereofcomprising a nucleotide sequence encoding at least one trehalosebiosynthetic enzyme such as, for example, a trehalose-6-phosphatesynthase or a trehalose-6-phosphate phosphatase under the control of apromoter capable of directing the expression of a nucleotide sequence inthe plastids of a plant; also including the seed for such a plant, whichseed is optionally treated (e.g., primed or coated) and/or packaged,e.g. placed in a bag or other container with instructions for use.

[0034] A plant comprising

[0035] a heterologous nuclear expression cassette or parts thereofpreferably comprising an inducible promoter, a tissue-specific promoteror a constitutive promoter, more preferably an inducible promoter, e.g.,a wound-inducible or chemically-inducible promoter, for example thetobacco PR-1a promoter, operably linked to a DNA sequence coding for atransactivator (preferably a transactivator not naturally occurring inplants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNApolymerase), said transactivator being optionally fused to a plastidtargeting sequence, e.g., a chloroplast targeting sequence (e.g., aplant expressible expression cassette as described above); and

[0036] a heterologous plastid expression cassette or parts thereofcomprising a transactivator-mediated promoter regulated by thetransactivator (e.g., the T7 promoter when the transactivator is T7 RNApolymerase) and operably linked to a nucleotide sequence encoding atleast one trehalose biosynthetic enzyme such as, for example, atrehalose-6-phosphate synthase;also including the seed for such a plant,which seed is optionally treated (e.g., primed or coated) and/orpackaged, e.g. placed in a bag or other container with instructions foruse.

[0037] The invention furthermore provides:

[0038] A plant comprising

[0039] a heterologous nuclear expression cassette or parts thereofpreferably comprising an inducible promoter, a tissue-specific promoteror a constitutive promoter, more preferably an inducible promoter, e.g.,a wound-inducible or chemically-inducible promoter, for example thetobacco PR-1a promoter, operably linked to a nucleotide sequenceencoding a transactivator (preferably a transactivator not naturallyoccurring in plants, preferably a RNA polymerase or DNA binding protein,e.g., T7 RNA polymerase), said transactivator being optionally fused toa plastid targeting sequence, e.g., a chloroplast targeting sequence(e.g., a plant expressible expression cassette as described above); and

[0040] a heterologous plastid expression cassette or parts thereofcomprising a transactivator-mediated promoter regulated by thetransactivator (e.g., the T7 promoter when the transactivator is T7 RNApolymerase) and operably linked to a nucleotide sequence encoding atrehalose-6-phosphate phosphatase;

[0041] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0042] The invention furthermore provides:

[0043] A plant comprising

[0044] a heterologous nuclear expression cassette or parts thereofpreferably comprising an inducible promoter, a tissue-specific promoteror a constitutive promoter, more preferably an inducible promoter, e.g.,a wound-inducible or chemically-inducible promoter, for example thetobacco PR-1a promoter, operably linked to a DNA sequence coding for atransactivator (preferably a transactivator not naturally occurring inplants, preferably a RNA polymerase or DNA binding protein, e.g., T7 RNApolymerase), said transactivator being optionally fused to a plastidtargeting sequence, e.g., a chloroplast targeting sequence (e.g., aplant expressible expression cassette as described above); and

[0045] a heterologous plastid expression cassette or parts thereofcomprising a transactivator-mediated promoter regulated by thetransactivator (e.g., the T7 promoter when the transactivator is T7 RNApolymerase) and operably linked to a nucleotide sequence encoding atrehalose-6-phosphate synthase and a transactivator-mediated promoterregulated by the transactivator (e.g., the T7 promoter when thetransactivator is T7 RNA polymerase) and operably linked to a nucleotidesequence encoding a trehalose-6-phosphate phosphatase;

[0046] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0047] In a further embodiment the invention encompasses expression ofnucleotide sequences encoding trehalose biosynthetic in the plastidunder the control of a promoter transcribed by a RNA polymerase normallypresent in plastids, such as a nuclear-encoded polymerase or aplastid-encoded polymerase. Such promoters are e.g., but not limited to,a cIpP promoter, a 16S r-RNA gene promoter, a psbA promoter or a rbcLpromoter.

[0048] The invention thus additionally provides:

[0049] A plant comprising

[0050] a heterologous plastid expression cassette or parts thereofpreferably comprising a promoter capable of expression of a nucleotidesequence encoding trehalose biosynthetic enzymes in plant plastids, forexample a promoter transcribed by a RNA polymerase normally present inplastids, such as a nuclear-encoded polymerase or a plastid-encodedpolymerase, operably linked to at least a nucleotide sequence encoding atrehalose biosynthetic enzyme such as, for example, atrehalose-6-phosphate synthase; also including the seed for such aplant, which seed is optionally treated (e.g., primed or coated) and/orpackaged, e.g. placed in a bag or other container with instructions foruse.

[0051] The invention furthermore provides:

[0052] A plant comprising

[0053] a heterologous plastid expression cassette or parts thereofpreferably comprising a promoter capable of expression of a nucleotidesequence encoding a trehalose biosynthetic enzyme in plant plastids, forexample a promoter transcribed by a RNA polymerase normally present inplastids, such as a nuclear-encoded polymerase or a plastid-encodedpolymerase, operably linked to a nucleotide sequence encoding atrehalose-6-phosphate phosphatase;

[0054] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0055] The invention furthermore provides:

[0056] A plant comprising

[0057] a heterologous plastid expression cassette or parts thereofpreferably comprising a promoter capable of expression of a nucleotidesequence encoding a trehalose biosynthetic enzyme in plant plastids, forexample a promoter transcribed by a RNA polymerase normally present inplastids, such as a nuclear-encoded polymerase or a plastid-encodedpolymerase, operably linked to a nucleotide sequence encoding atrehalose-6-phosphate synthase and a promoter transcribed by a RNApolymerase normally present in plastids, such as a nuclear-encodedpolymerase or a plastid-encoded polymerase, operably linked to anucleotide sequence encoding a trehalose-6-phosphate phosphatase;

[0058] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0059] In a further embodiment, the invention encompasses the expressionfrom a single promoter of two or more genes in plant plastids in anoperon-like polycistronic gene. In a preferred embodiment, anoperon-like polycistronic gene comprises the two or more genes, e.g.genes comprising a nucleotide sequence encoding a trehalose biosyntheticenzyme, operably linked to a promoter capable of directing theexpression of operon-like polycistronic gene in plastids and is insertedinto the plastid genome. In a preferred embodiment, the operon-likepolycistronic gene comprises an intervening DNA sequence between twogenes in the operon-like polycistronic gene, preferably a DNA sequencenot present in the plastid genome. In another preferred embodiment, theintervening DNA sequence is derived from the 5′ untranslated (UTR)region of a non-eukaryotic gene, preferably a viral 5′UTR, preferably a5′UTR derived from a bacterial phage, such as a T7, T3 or SP6 phage. Ina preferred embodiment, the DNA sequence is modified to prevent theformation of secondary structures that inhibit or repress translation ofthe gene located immediately downstream of the intervening DNA sequence.In a preferred embodiment, the expression, preferably the translation,of genes located immediately downstream of the intervening DNA sequenceis increased.

[0060] The invention thus furthermore provides:

[0061] A plant comprising

[0062] a heterologous nuclear expression cassette or parts thereofpreferably comprising an inducible promoter, a tissue-specific promoteror a constitutive promoter, more preferably an inducible promoter, e.g.,a wound-inducible or chemically-inducible promoter, for example thetobacco PR-1a promoter, operably linked to a nucleotide sequenceencoding a transactivator (preferably a transactivator not naturallyoccurring in plants, preferably a RNA polymerase or DNA binding protein,e.g., T7 RNA polymerase), said transactivator being optionally fused toa plastid targeting sequence, e.g., a chloroplast targeting sequence(e.g., a plant expressible expression cassette as described above); and

[0063] a heterologous plastid expression cassette or parts thereofcomprising a transactivator-mediated promoter regulated by thetransactivator (e.g., the T7 promoter when the transactivator is T7 RNApolymerase) and operably linked to an operon-like polycistronic genecomprising at least one gene comprising a nucleotide sequence encoding atrehalose biosynthetic enzyme. In a preferred embodiment, theoperon-like polycistronic gene comprises one gene comprising anucleotide sequence encoding a trehalose phosphate synthase and one geneencoding a nucleotide sequence encoding a trehalose phosphatephosphatase. In a preferred embodiment, the operon-like polycistronicgene comprises an intervening DNA sequence between two genes in theoperon-like polycistronic gene, preferably a DNA sequence not present inthe plastid genome. In a preferred embodiment, the DNA sequence isderived from the 5′ untranslated (UTR) region of a non-eukaryotic gene,preferably a viral 5′UTR, preferably a 5′UTR derived from a bacterialphage, such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNAsequence is modified to prevent the formation of secondary structuresthat inhibit or repress translation of the gene located immediatelydownstream of the intervening DNA sequence. In a preferred embodiment,the expression, preferably the translation, of genes located immediatelydownstream of the intervening DNA sequence is increased.

[0064] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0065] The invention furthermore provides:

[0066] A plant comprising

[0067] a heterologous nuclear expression cassette or parts thereofpreferably comprising a promoter capable of expression of a nucleotidesequence encoding a trehalose biosynthetic enzyme in plant plastids, forexample a promoter transcribed by a RNA polymerase normally present inplastids, such as a nuclear-encoded polymerase or a plastid-encodedpolymerase, operably linked to an operon-like polycistronic genecomprising at least one gene comprising a nucleotide sequence encoding atrehalose biosynthetic enzyme. In a preferred embodiment, theoperon-like polycistronic gene comprises one gene comprising anucleotide sequence encoding a trehalose phosphate synthase and one geneencoding a nucleotide sequence encoding a trehalose phosphatephosphatase. In a preferred embodiment, the operon-like polycistronicgene comprises an intervening DNA sequence between two genes in theoperon-like polycistronic gene, preferably a DNA sequence not present inthe plastid. In a preferred embodiment, the DNA sequence is derived fromthe 5′ untranslated (UTR) region of a noneukaryotic gene, preferably aviral 5′UTR, preferably a 5′UTR derived from a bacterial phage, such asa T7, T3 or SP6 phage. In a preferred embodiment, the DNA sequence ismodified to prevent the formation of secondary structures that inhibitor repress translation of the gene located immediately downstream of theintervening DNA sequence. In a preferred embodiment, the expression,preferably the translation, of genes located immediately downstream ofthe intervening DNA sequence is increased.

[0068] also including the seed for such a plant, which seed isoptionally treated (e.g., primed or coated) and/or packaged, e.g. placedin a bag or other container with instructions for use.

[0069] The invention furthermore provides:

[0070] A plant expressible expression cassette preferably comprising aninducible promoter, e.g., a wound-inducible or chemically-induciblepromoter, for example the tobacco PR-1a promoter, operably linked to anucleotide sequence encoding a transactivator (preferably atransactivator not naturally occurring in plants, preferably a RNApolymerase or DNA binding protein, e.g., T7 RNA polymerase), saidtransactivator being fused to a plastid targeting sequence, e.g., achloroplast targeting sequence;

[0071] a vector comprising such a plant expressible cassette; and

[0072] a plant transformed with such a vector or a transgenic plant thegenome of which comprises such a plant expressible expression cassette.

[0073] The invention also provides:

[0074] A heterologous plastid expression cassette comprising atransactivator-mediated promoter regulated by the transactivator (e.g.,the T7 promoter when the transactivator is T7 RNA polymerase) andoperably linked to a nucleotide sequence encoding at least one trehalosebiosynthetic enzyme such as, for example, a trehalose-6-phosphatesynthase and/or a trehalose 6-phosphate phosphatase.

[0075] The invention also provides:

[0076] A heterologous plastid expression cassette comprising a promotertranscribed by a RNA polymerase normally present in plastids, such as anuclear-encoded polymerase or a plastid-encoded polymerase and operablylinked to a nucleotide sequence encoding at least one trehalosebiosynthetic enzyme such as, for example, a trehalose-6-phosphatesynthase and/or trehalose 6-phosphate phosphatase.

[0077] The invention also provides:

[0078] A heterologous plastid expression cassette comprising a promotercapable of expression of a trehalose biosynthetic gene in plantplastids, for example a promoter transcribed by a RNA polymerasenormally present in plastids, such as a nuclear-encoded polymerase or aplastid-encoded polymerase, or a transactivator-mediated promoterregulated by the transactivator (e.g., the T7 promoter when thetransactivator is T7 RNA polymerase), operably linked to an operon-likepolycistronic gene comprising nucleotide sequences encoding bothtrehalose biosynthetic enzymes. In a preferred embodiment, theoperon-like polycistronic gene comprises an intervening DNA sequencebetween two genes in the operon-like polycistronic gene, preferably aDNA sequence not present in the plastid genome. In a preferredembodiment, the DNA sequence is comprises a portion of the 5′untranslated (UTR) region of a non-eukaryotic gene, preferably a viral5′UTR, preferably a 5′UTR derived from a bacterial phage, such as a T7,T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modifiedto prevent the formation of secondary structures that inhibit or repressthe translation of the gene located immediately downstream of theintervening DNA sequence. In a preferred embodiment, the expression,preferably the translation, of genes located immediately downstream ofthe intervening DNA sequence is increased.

[0079] The invention also comprises:

[0080] A method of producing a plant as described above comprising

[0081] pollinating a plant comprising a heterologous plastid expressioncassette or parts thereof comprising a transactivator-mediated promoterregulated and operably linked to a nucleotide sequence of interest, butpreferably a nucleotide sequence encoding at least one trehalosebiosynthetic enzyme such as, for example a trehalose-6-phosphatesynthase and/or a trehalose 6-phosphate phosphatase with pollen from aplant comprising a heterologous nuclear expression cassette or partsthereof comprising an inducible promoter, a tissue-specific promoter ora constitutive promoter, more preferably an inducible promoter, operablylinked to a nucleotide sequence encoding a transactivator capable ofregulating said transactivator-mediated promoter;

[0082] recovering seed from the plant thus pollinated; and

[0083] cultivating a plant as described above from said seed.

[0084] The invention further provides:

[0085] A method for producing trehalose in a plant by expressing atleast one heterologous nucleotide sequence encoding a trehalosebiosynthetic enzyme under the control of any one of the promotersdescribed above, for example an inducible promoter, e.g., a woundinducible or chemically inducible promoter, in the nuclear genome ofsaid plant or by expressing at least one heterologous nucleotidesequence encoding a trehalose biosynthetic enzyme in the plastids ofsaid plant under the control of a promoter capable of expressing saidnucleotide sequence in the plastids of said plant or in any one of theexpression cassettes described above.

[0086] A method for protecting a plant against drought, high salinity,osmotic stress and temperature extremes by expressing in said plant atleast one nucleotide sequence encoding a trehalose biosynthetic enzymefrom the nuclear genome of said plant under the control of an induciblepromoter e.g., a wound inducible or chemically inducible promoter, orfrom the plastid genome of said plant under the control of a promotercapable of expressing said nucleotide sequence in the plastids of saidplant.

[0087] A method for increasing storage properties of harvested plants byexpressing in said plant at least one nucleotide sequence encoding atrehalose biosynthetic enzyme from the nuclear genome of said plantunder the control of an inducible promoter e.g., a wound inducible orchemically inducible promoter, or from the plastid genome of said plantunder the control of a promoter capable of expressing said nucleotidesequence in the plastids of said plant.

[0088] A method for improving shelf-life of fruits and vegetables, andpreserving flowers by expressing in said fruits, vegetables and flowersat least one nucleotide sequence encoding a trehalose biosyntheticenzyme from the nuclear genome of said plant under the control of aninducible promoter e.g., a wound inducible or chemically induciblepromoter, or from the plastid genome of said plant under the control ofa promoter capable of expressing said nucleotide sequence in theplastids of said plant.

[0089] A method for stabilizing proteins, preferably transgenicproteins, expressed in transgenic plants by expressing in said plant atleast one nucleotide sequence encoding a trehalose biosynthetic enzymefrom the nuclear genome of said plant under the control of an induciblepromoter e.g., a wound inducible or chemically inducible promoter, orfrom the plastid genome of said plant under the control of a promotercapable of expressing said nucleotide sequence in the plastids of saidplant.

[0090] The present invention further provides:

[0091] A method of expressing two or more genes from a single promoterin the plastids of a plant comprising introducing into the plastidgenome of said plant a operon-like polycistronic gene comprising saidtwo or more genes operably linked to a promoter capable of expressingsaid operon-like polycistronic gene in the plastids of said plant,wherein said operon-like polycistronic gene further comprises anintervening DNA sequence between two genes. In a preferred embodiment, aDNA sequence not present in the plastid genome. In a preferredembodiment, the DNA sequence is comprises a portion of the 5′untranslated (UTR) region of a non-eukaryotic gene, preferably a viral5′UTR, preferably a 5′UTR derived from a bacterial phage, such as a T7,T3 or SP6 phage. In a preferred embodiment, the DNA sequence is modifiedto prevent the formation of secondary structures that inhibit or repressthe translation of the gene located immediately downstream of theintervening DNA sequence. In a preferred embodiment, the expression,preferably the translation, of genes located immediately downstream ofthe intervening DNA sequence is increased.

[0092] In a preferred embodiment, the operon-like polycistronic genecomprises at least one gene comprising a nucleotide sequence encoding atrehalose biosynthetic gene. In another preferred embodiment, theoperon-like polycistronic gene comprises a gene comprising a nucleotidesequence encoding a trehalose phosphate synthase and a gene comprising anucleotide sequence encoding a trehalose phosphate phosphatase.

DEFINITIONS

[0093] In order to ensure a clear and consistent understanding of thespecification and the claims, the following definitions are provided:

[0094] “Drought resistance” is the trait of a transgenictrehalose-producing plant to sustain prolonged periods of time receivingless water than a wild-type (nontransgenic) plant would normally requireor without being watered, and without showing the same degree of wiltingof its leaves or other characteristics of dessication that appear in awild-type plant grown under the same conditions.

[0095] “Gene” as used herein comprises a nucleotide sequence optionallyoperably linked to DNA sequences preceding or following the nucleotidesequence. The nucleotide sequence is typically transcribable into RNA,such as e.g. mRNA (sense RNA or antisense RNA), rRNA, tRNA or snRNA. Anucleotide sequence in a gene optionally comprises a coding sequence,which can be translated into a polypeptide. Examples of DNA sequencespreceding or following the nucleotide sequence are 5′ and 3′untranslated sequences, termination signals and ribosome binding sites(rbs), or portions thereof. Further elements that may also be present ina gene are, for example, introns.

[0096] “Expression cassette” as used herein means a DNA constructdesigned so that a nucleotide sequence inserted herein can betranscribed and, optionally translated, in an appropriate host cell. Theexpression cassette typically comprises regulatory elements, such as apromoter capable of directing expression of the nucleotide sequenceoperably linked to the nucleotide sequence, which is itself optionallyoperably linked to 3′ sequences, such as 3′ regulatory sequences ortermination signals. The expression cassette also may comprisessequences required for proper translation of a coding sequence comprisedin the nucleotide sequence. The nucleotide sequence usually comprisesthe coding sequence of a protein but may also code for a functional RNAof interest, for example antisense RNA or a nontranslated RNA that, inthe sense or antisense direction, inhibits expression of a particulargene, e.g., antisense RNA. The expression cassette comprising thenucleotide sequence of interest may be polycistronic, meaning that atleast one of its components is heterologous with respect to at least oneof its other components. The expression cassette may also be one whichis naturally occurring but has been obtained in a recombinant formuseful for heterologous expression. Typically, however, the expressioncassette is heterologous with respect to the host, i.e., the particularDNA sequence of the expression cassette does not occur naturally in thehost cell and must have been introduced into the host cell or anancestor of the host cell. The expression of the nucleotide sequence inthe expression cassette may be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, such as a plant, the promoter canalso be specific to a particular tissue or organ or stage ofdevelopment. A nuclear expression cassette is usually inserted into thenuclear genome of a plant and is capable of directing the expression ofa particular nucleotide sequence from the nuclear genome of said plant.A plastid expression cassette is usually inserted in to the plastidgenome of a plant and is capable of directing the expression of aparticular nucleotide sequence from the plastid genome of said plant,for example a promoter transcribed by a RNA polymerase normally presentin plastids, such as a nuclear-encoded polymerase or a plastid-encodedpolymerase, or a transactivator-mediated promoter. A plastid expressioncassette as described herein may optionally comprise an operon-likepolycistronic gene.

[0097] “Regulatory elements” refer to DNA sequences involved in theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operably linked to the nucleotide sequence of interest, and mayalso include 5′ and 3′ untranslated regions (UTR) or terminationsignals. They also typically encompass sequences required for propertranslation of the nucleotide sequence, such as, in the case ofexpression in plastids, ribosome binding sites (rbs).

[0098] “Heterologous” as used herein means “of different natural origin”or represents a nonnatural state. For example, if a host cell istransformed with a nucleotide sequence derived from another organism,particularly from another species, that nucleotide sequence isheterologous with respect to that host cell and also with respect todescendants of the host cell which carry that gene. Similarly,heterologous refers to a nucleotide sequence derived from and insertedinto the same natural, original cell type, but which is present in anon-natural state, e.g. a different copy number, or under the control ofdifferent regulatory elements. A transforming nucleotide sequence maycomprise a heterologous coding sequence, or heterologous regulatoryelements. Alternatively, the transforming nucleotide sequence may becompletely heterologous or may comprise any possible combination ofheterologous and endogenous nucleic acid sequences.

[0099] “Expression” refers to the transcription and/or translation of anucleotide sequence, for example an endogenous gene or a heterolousgene, in a host organism, e.g. microbes or plants. In the case ofantisense constructs, for example, expression may refer to thetranscription of the antisense DNA only

[0100] An “operon-like polycistronic gene” comprises two or more genesof interest under control of a single promoter capable of directing theexpression of such operon-like polycistronic gene in plant plastids.Every gene in a operon-like polycistronic gene optionally comprises aribosome binding site (rbs) operably linked to the 5′ end of thenucleotide sequence. Preferably each rbs in the operon-likepolycistronic gene is different. The operon-like polycistronic gene alsotypically comprises a 5′ UTR operably linked to the 5′ end of the rbs ofthe first gene in the operon-like polycistronic gene and a 3′ UTRoperably linked to the 3′ end of the last gene in the operon-likepolycistronic gene. Two genes in a operon-like polycistronic gene mayalso comprise several nucleic acids which overlap between the two genes.

[0101] “Homoplastidic” refers to a plant, plant tissue or plant cellwherein all of the plastids are genetically identical. This is thenormal state in a plant when the plastids have not been transformed,mutated, or otherwise genetically altered. In different tissues orstages of development, the plastids may take different forms, e.g.,chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts, and soforth.

[0102] “Marker gene”: a gene encoding a selectable or screenable trait.

[0103] “Inducible Promoter”: An “inducible promoter” is a promoter whichinitiates transcription only when the plant is exposed to someparticular external stimulus, as distinguished from constitutivepromoters or promoters specific to a specific tissue or organ or stageof development. Particularly preferred for the present invention arechemically-inducible promoters and wound-inducible promoters. Chemicallyinducible promoters include plant-derived promoters, such as thepromoters in the systemic acquired resistance pathway, for example thePR promoters, e.g., the PR-1, PR-2, PR-3, PR-4, and PR-5 promoters,especially the tobacco PR-1a promoter and the Arabidopsis PR-1 promoter,which initiate transcription when the plant is exposed to BTH andrelated chemicals. See U.S. Pat. No. 5,614,395, incorporated herein byreference, and WO 98/03536, incorporated herein by reference.Chemically-inducible promoters also include receptor-mediated systems,e.g., those derived from other organisms, such as steroid-dependent geneexpression, copper-dependent gene expression, tetracycline-dependentgene expression, and particularly the expression system utilizing theUSP receptor from Drosophila mediated by juvenile growth hormone and itsagonists, described in EP-A 0 859 851, incorporated herein by reference,as well as systems utilizing combinations of receptors, e.g., asdescribed in EP-A 0 813 604, incorporated herein by reference. Woundinducible promoters include promoters for proteinase inhibitors, e.g.,the proteinase inhibitor II promoter from potato, and otherplant-derived promoters involved in the wound response pathway, such aspromoters for polyphenyl oxidases, LAP and TD. See generally, C. Gatz,“Chemical Control of Gene Expression”, Annu. Rev. Plant Physiol. PlantMol. Biol. (1997) 48: 89-108, the contents of which are incorporatedherein by reference.

[0104] “Operably linked to/associated with”: a DNA sequence, for examplecomprising a regulatory element, is said to be “operably linked to” or“associated with” a nucleotide sequence if the two sequences aresituated such that the DNA sequence affects expression of the nucleotidesequence.

[0105] “Phenotypic trait”: a detectable property resulting from theexpression of one or more genes.

[0106] “Plant”: A “plant” refers to any plant or part of a plant at anystage of development. In some embodiments of the invention, the plantsmay be lethally wounded to induce expression or may be induced toexpress lethal levels of a desired protein, and so the term “plant” asused herein is specifically intended to encompass plants and plantmaterial which have been seriously damaged or killed, as well as viableplants, cuttings, cell or tissue cultures, and seeds. Preferably, plantsof the present invention are distinguished in that they aredevelopmentally normal up to the point of induction of the trehalosebiosynthetic gene.

[0107] “Plant cell”: a structural and physiological unit of the plant,comprising a protoplast and a cell wall. The plant cell may be in formof an isolated single cell or a cultured cell, or as a part of higherorganized unit such as, for example, a plant tissue, or a plant organ.

[0108] “Plant material”: refers to leaves, stems, roots, flowers orflower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, eggcells, zygotes, embryos, seeds, plastids, mitochondria, cuttings, cellor tissue cultures, or any other part or product of a plant.

[0109] “Progeny” as used herein comprises all the subsequent generationsobtained by self-pollination or out-crossing of a plant of the presentinvention.

[0110] “Promoter”: a DNA sequence that initiates transcription of anassociated DNA sequence. The promoter region may also include elementsthat act as regulators of gene expression such as activators, enhancers,and/or repressors.

[0111] “Protoplast”: isolated plant cell where the cell wall has beentotally or partially removed.

[0112] “Recombinant DNA molecule”: a combination of DNA sequences thatare joined together using recombinant DNA technology.

[0113] “Recombinant DNA technology”: procedures used to join togetherDNA sequences as described, for example, in Sambrook et al., 1989, ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

[0114] “Screenable marker gene”: a gene whose expression does not confera selective advantage to a transformed cell, but whose expression makesthe transformed cell phenotypically distinct from untransformed cells.

[0115] “Selectable marker gene”: a gene whose expression in a plant cellgives the cell a selective advantage. The selective advantage possessedby the cells transformed with the selectable marker gene may be due totheir ability to grow in the presence of a negative selective agent,such as an antibiotic or a herbicide, compared to the growth ofnon-transformed cells. The selective advantage possessed by thetransformed cells, compared to non-transformed cells, may also be due totheir enhanced or novel capacity to utilize an added compound as anutrient, growth factor or energy source. Selectable marker gene alsorefers to a gene or a combination of genes whose expression in a plantcell gives the cell both, a negative and a positive selective advantage.

[0116] In its broadest sense, the term “substantially similar”, whenused herein with respect to a nucleotide sequence, means a nucleotidesequence corresponding to a reference nucleotide sequence, wherein thecorresponding sequence encodes a polypeptide having substantially thesame structure and function as the polypeptide encoded by the referencenucleotide sequence, e.g. where only changes in amino acids notaffecting the polypeptide function occur. Desirably the substantiallysimilar nucleotide sequence encodes the polypeptide encoded by thereference nucleotide sequence. The percentage of identity between thesubstantially similar nucleotide sequence and the reference nucleotidesequence desirably is at least 80%, more desirably at least 85%,preferably at least 90%, more preferably at least 95%, still morepreferably at least 99%. Sequence comparisons are carried out using aSmith-Waterman sequence alignment algorithm (see e.g. Waterman, M. S.Introduction to Computational Biology: Maps, sequences and genomes.Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or athttp://www-hto.usc.edu/software/seqaln/index.html). The locals program,version 1.16, is used with following parameters: match: 1, mismatchpenalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. Anucleotide sequence “substantially similar” to reference nucleotidesequence hybridizes to the reference nucleotide sequence in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSc, 0.1% SDSat 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSc, 0.1% SDS at 50°C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 0.1×SSc, 0.1% SDS at 50° C., morepreferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 0.1×SSc, 0.1% SDS at 65° C.

[0117] The term “substantially similar”, when used herein with respectto a protein, means a protein corresponding to a reference protein,wherein the protein has substantially the same structure and function asthe reference protein, e.g. where only changes in amino acids notaffecting the polypeptide function occur. When used for a protein or anamino acid sequence the percentage of identity between the substantiallysimilar and the reference protein or amino acid sequence desirably is atleast 80%, more desirably 85%, preferably at least 90%, more preferablyat least 95%, still more preferably at least 99%.

[0118] “Transactivator”: A “transactivator” is a protein which, byitself or in combination with one or more additional proteins, iscapable of causing transcription of a coding region under control of acorresponding transactivator-mediated promoter. Examples oftransactivator systems include phage T7 gene 10 promoter, thetranscriptional activation of which is dependent upon a specific RNApolymerase such as the phage T7 RNA polymerase. The transactivator istypically an RNA polymerase or DNA binding protein capable ofinteracting with a particular promoter to initiate transcription, eitherby activating the promoter directly or by inactivating a repressor gene,e.g., by suppressing expression or accumulation of a repressor protein.The DNA binding protein may be a chimeric protein comprising a bindingregion (e.g., the GAL4 binding region) linked to an appropriatetranscriptional activator domain. Some transactivator systems may havemultiple transactivators, for example promoters which require not only apolymerase but also a specific subunit (sigma factor) for promotorrecognition, DNA binding, or transcriptional activation. Thetransactivator is preferably heterologous with respect to the plant.

[0119] “Transformation:” Introduction of a nucleotide sequence into acell. In particular, the stable integration of a DNA molecule into thegenome of an organism of interest.

[0120] “Trehalose biosynthetic enzymes” are polypeptides involved in thebiosynthesis of trehalose from glucose, e.g., as described herein,particularly trehalose-6-phosphate synthase which catalyses thecondensation of UDP-glucose and glucose-6-phosphate intotrehalose-6phosphate or trehalose-6-phosphate phosphatase whichphosphorylates trehalose-6-phosphate to trehalose. The nucleotidesequences encoding the trehalose biosynthetic enzymes are comprised intrehalose biosynthetic genes.

[0121] “Trehalose” is a D-glucopyranosyl-[1,1]-D-glucopyranoside. Theprefered form of trehalose in the present invention is a,a-trehalose(a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside).

DETAILLED DESCRIPTION OF THE INVENTION

[0122] The present invention also encompasses cells comprising a DNAmolecule of the present invention, wherein the DNA molecule is not inits natural cellular environment. In a preferred embodiment, such cellsare plant cells. In another prefered embodiment, a DNA molecule of thepresent invention is expressible in such cells and is comprised in anexpression cassette which allow their expression in such cells. In apreferred embodiment, the expression cassette is stably integrated intothe DNA of such host cell. In another preferred embodiment, theexpression cassette is comprised in a vector, which is capable ofreplication in the cell and remains in the cell as an extrachromosomalmolecule.

[0123] In the present invention, trehalose biosynthetic genes from E.coli are preferably used, but trehalose biosynthetic genes from otherorganisms including but not limited to yeast, other lower organisms orhigher organisms, such as plants, are suitable. For example, the yeastTPS1, TSL1 or TSL2 genes (U.S. Pat. No. 5,792,921), the Arabidopsistrehalose synthase gene (TPS1, accession number Y08568, Blazquez et al.Plant J (1998) 13:685-9), Arabidopsis trehalose phosphate phosphatases(Vogel et al. Plant J (1998) 13:673-83) or a Selaginella lepidophyllagene (accession number U96736).

[0124] The present invention also encompasses a plant comprising theplant cells described above. In a further embodiment, the DNA moleculesof the present invention are expressible in the plant, and expression ofany one of the DNA molecules of the present invention or of a functionalportion or derivative thereof in transgenic plants confers production oftrehalose and leads to drought-resistance, increased storage propertiesof the harvested plant, improved food quality, e.g. shelf-life of fruitsor vegetables derived from said plant, improved longevity of flowers,stabilization of proteins expressed in said plant, preferably transgenicproteins, or high levels of trehalose useful for industrial production,and other characteristics as described herein. The present inventiontherefore also encompasses transgenic plants which express trehalose dueto the expression of any one of the DNA molecules of the presentinvention or of a functional portion or derivative thereof.

[0125] Plants transformed in accordance with the present invention maybe monocots or dicots and include, but are not limited to, maize, wheat,barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear,quince, melon, plum, cherry, peach, nectarine, apricot, strawberry,grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana,soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed,clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant,cucumber, Arabidopsis thaliana, and woody plants such as coniferous anddeciduous trees.

[0126] Preferred are monocot plants selected from the group consistingof maize, wheat barley, rye, sorghum, and rice. Further preferred aredicot plants selected from the group consisting of chicory, lettuce,cabbage, cauliflower, broccoli, pepper, squash, pumpkin, zucchini,melon, soybean, tomato, sugarcane, sugarbeet, sunflower, rapeseed,cotton, and alfalfa. Preferred groups of plants are plants producingvegetable, fruits and flowers.

[0127] Once a desired nucleotide sequence has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques, e.g. byrecurrent selection breeding, like backcrossing. In this case, therecurrent parent in which the desired transgene is to be introgressed isfirst crossed to the non-recurrent parent that carries the transgene inquestion. The progeny of this cross is then mated back to the recurrentparent followed by selection in the resultant progeny for the transgeneto be transferred from the nonrecurrent parent. After three, preferablyfour, more preferably five or more generations of backcrosses with therecurrent parent with selection for the transgene, the progeny will beheterozygous for the transgene being transferred, but will be like therecurrent parent for most or almost all other genes.

[0128] For their expression in transgenic plants, the DNA molecules mayrequire modification and optimization, particularly when the DNAmolecules are of prokaryotic origin. It is known in the art that allorganisms have specific preferences for codon usage, and the codons inthe nucleotide sequence comprised in the DNA molecules of the presentinvention can be changed to conform with specific plant preferences,while maintaining the amino acids encoded thereby. Furthermore, highexpression in plants is best achieved from coding sequences which haveat least 35% GC content, and preferably more than 45%. Nucleotidesequences which have low GC contents may express poorly due to theexistence of ATTTA motifs which may destabilize messages, and AATAAAmotifs which may cause inappropriate polyadenylation. Although preferredgene sequences may be adequately expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)). In addition,the nucleotide sequences are screened for the existence of illegitimatesplice sites which cause message truncation. All changes required to bemade within the nucleotide sequences such as those described above aremade using well known techniques of site directed mutagenesis, PCR, andsynthetic gene construction using the methods described in the publishedpatent applications EP 0 385 962 (to Monsanto), EP 0 359 472 (toLubrizol), and WO 93/07278 (to Ciba-Geigy).

[0129] For efficient initiation of translation, sequences adjacent tothe initiating methionine may require modification. For example, theycan be modified by the inclusion of sequences known to be effective inplants. Joshi has suggested an appropriate consensus for plants (NAR 15:6643-6653 (1987)) and Clontech suggests a further consensus translationinitiator (1993/1994 catalog, page 210). These consensuses are suitablefor use with the nucleotide sequences of this invention. The sequencesare incorporated into constructions comprising the nucleotide sequence,up to and including the ATG (whilst leaving the second amino acidunmodified), or alternatively up to and including the GTC subsequent tothe ATG (with the possibility of modifying the second amino acid of thetransgene).

[0130] In transgenic plants, the DNA molecules of the present invention,for example trehalose biosynthetic genes or genes encoding atransactivator, are driven by a promoter shown to be functional inplants. The choice of promoter will vary depending on the temporal andspatial requirements for expression, and also depending on the targetspecies. Although many promoters from dicotyledons have been shown to beoperational in monocotyledons and vice versa, ideally dicotyledonouspromoters are selected for expression in dicotyledons, andmonocotyledonous promoters for expression in monocotyledons. However,there is no restriction to the provenance of selected promoters; it issufficient that they are operational in driving the expression of theDNA molecules in the desired cell.

[0131] Preferred promoters which are expressed constitutively includethe CaMV 35S and 19S promoters, promoters from genes encoding actin orubiquitin, and promoters derived from Agrobacterium, for examplesynthetic promoters as described in PCT/US94/12946. The DNA molecules ofthis invention, however, are preferably expressed under the regulationof promoters which are chemically regulated. This enables the trehaloseto be synthesized only when the crop plants are treated with theinducing chemicals, thereby avoiding developmental abnormalities in theyoung plants. Preferred technology for chemical induction of geneexpression is detailed in the published application EP 0 332 104 (toCiba-Geigy) and U.S. Pat. No. 5,614,395. A preferred promoter forchemical induction is the tobacco PR-1a promoter.

[0132] A second preferred category of inducible promoters is that whichis wound inducible, permitting expression of the trehalose biosyntheticenzymes when the plant is injured, for example at harvest, or in silageor other processing. Numerous promoters have been described which areexpressed at wound sites. Preferred promoters of this kind include thosedescribed by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu etal. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792(1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warneret al. Plant J. 3: 191-201 (1993).

[0133] Preferred tissue specific expression patterns include greentissue specific, root specific, stem specific, and flower specific.Promoters suitable for expression in green tissue include many whichregulate genes involved in photosynthesis and many of these have beencloned from both monocotyledons and dicotyledons. A preferred promoteris the maize PEPC promoter from the phosphoenol carboxylase gene(Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)). A preferredpromoter for root specific expression is that described by de Framond(FEBS 290: 103-106 (1991); EP 0 452 269 to Ciba-Geigy) and a furtherpreferred root-specific promoter is that from the T-1 gene provided bythis invention. A preferred stem specific promoter is that described inU.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression ofthe maize trpA gene.

[0134] In addition to the selection of a suitable promoter,constructions for expression of the protein in plants optionally requirean appropriate transcription terminator to be attached downstream of theheterologous nucleotide sequence. Several such terminators are availableand known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any availableterminator known to function in plants can be used in the context ofthis invention. Numerous other sequences can be incorporated intoexpression cassettes for the DNA molecules of this invention. Theseinclude sequences which have been shown to enhance expression such asintron sequences (e.g. from Adh 1 and bronze1) and viral leadersequences (e.g. from TMV, MCMV and AMV).

[0135] It may be preferable to target expression of the DNA molecules todifferent cellular localizations in the plant. In some cases,localization in the cytosol may be desirable, whereas in other cases,localization in some subcellular organelle may be prefered. Subcellularlocalization of transgene encoded enzymes can be undertaken usingtechniques well known in the art. Typically, the DNA encoding the targetpeptide from a known organelle-targeted gene product is manipulated andfused upstream of the nucleotide sequence. Many such target sequencesare known for the chloroplast and their functioning in heterologousconstructions has been shown. A preferred class of targeting sequencesare the vacuole targeting sequences, e.g., as found on plant chitinasesand proteases.

[0136] Vectors suitable for plant transformation are described elsewherein this specification. For Agrobacterium-mediated transformation, binaryvectors or vectors carrying at least one T-DNA border sequence aresuitable, whereas for direct gene transfer any vector is suitable andlinear DNA containing only the construction of interest may bepreferred. In the case of direct gene transfer, transformation with asingle DNA species or co-transformation can be used (Schocher et al.Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer andAgrobacterium-mediated transfer, transformation is usually (but notnecessarily) undertaken with a selectable marker which may provideresistance to an antibiotic (kanamycin, hygromycin or methatrexate) or aherbicide (basta). The choice of selectable marker is not, however,critical to the invention.

[0137] In another preferred embodiment, the DNA molecules of thisinvention are directly transformed into the plastid genome. Plastidtransformation technology is described extensively in U.S. Pat. Nos.5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos.WO 95/16783 and WO 97/32977; and in McBride et al., Proc. Natl. Acad.Sci. USA 91: 7301-7305 (1994), all of which are incorporated herein byreference. Plastid transformation via biolistics was achieved initiallyin the unicellular green alga Chlamydomonas reinhardtii (Boynton et al.(1988) Science 240: 1534-1537, incorporated herein by reference) andthis approach, using selection for cis-acting antibiotic resistance loci(spectinomycin/streptomycin resistance) or complementation ofnon-photosynthetic mutant phenotypes, was soon extended to Nicotianatabacum (Svab et al. (1990) Proc. Natl. Acad. Sci. USA. 87: 8526-8530,incorporated herein by reference).

[0138] The basic technique for tobacco plastid transformation involvesthe particle bombardment of leaf or callus tissue or PEG-mediated uptakeof plasmid DNA in protoplasts with regions of cloned plastid DNAflanking a selectable antibiotic resistance marker. The 1 to 1.5 kbflanking regions, termed targeting sequences, facilitate homologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the 156 kb tobacco plastid genome.Initially, point mutations in the plastid 16S rDNA and rps12 genesconferring resistance to spectinomycin and/or streptomycin were utilizedas selectable markers for transformation (Svab, Z., Hajdukiewicz, P.,and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub,J. M., and Maliga, P. (1992) Plant Cell 4, 39-45, incorporated herein byreference). This resulted in stable homoplasmic transformants at afrequency of approximately one per 100 bombardments of target leaves.The presence of cloning sites between these markers allowed creation ofa plastid targeting vector for introduction of foreign genes (Staub, J.M., and Maliga, P., EMBO J. 12: 601-606 (1993), incorporated herein byreference). Substantial increases in transformation frequency wereobtained by replacement of the recessive rRNA or r-protein antibioticresistance genes with a dominant selectable marker, the bacterial aadAgene encoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P. (1993)Proc. Natl. Acad. Sci. USA 90, 913-917, incorporated herein byreference). Previously, this marker had been used successfully forhigh-frequency transformation of the plastid genome of the green algaChlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. AcidsRes. 19, 4083-4089, incorporated herein by reference). Recently, plastidtransformation of protoplasts from tobacco and the moss Physcomitrellapatens has been attained using polyethylene glycol (PEG) mediated DNAuptake (O'Neill et al. (1993) Plant J. 3: 729-738; Koop et al. (1996)Planta 199: 193-201, both of which are incorporated herein byreference). Both particle bombardment and protoplast transformation areappropriate in the context of the present invention.

[0139] A DNA molecule of the present invention is inserted into aplastid expression cassette comprising a promoter capable of expressingthe DNA molecule in plant plastids. A preferred promoter capable ofexpression in a plant plastid is a promoter isolated from the 5′flanking region upstream of the coding region of a plastid gene, whichmay come from the same or a different species, and the native product ofwhich is typically found in a majority of plastid types including thosepresent in non-green tissues. Gene expression in plastids differs fromnuclear gene expression and is related to gene expression in prokaryotes(described in Stern et al. (1997) Trends in Plant Sciences 2: 308-315,incorporated herein by reference). Plastid promoters generally containthe -35 and -10 elements typical of prokaryotic promoters and someplastid promoters are recognized by a E. coli-like RNA polymerase mostlyencoded in the plastid genome and are called PEP (plastid-encoded RNApolymerase) promoters while other plastid promoters are recognized by anuclear-encoded RNA polymerase (NEP promoters). Both types of plastidpromoters are suitable for the present invention. Examples of plastidpromoters are promoters of clpP genes, such as the tobacco clpP genepromoter (WO 97/06250, incorporated herein by reference) and theArabidopsis clpP gene promoter (U.S. application Ser. No. 09/038,878,incorporated herein by reference). Another promoter that is capable ofexpressing a DNA molecule in plant plastids comes from the regulatoryregion of the plastid 16S ribosomal RNA operon (Harris et al.,Microbiol. Rev. 58:700-754 (1994), Shinozaki et al., EMBO J. 5:2043-2049(1986), both of which are incorporated herein by reference). Otherexamples of promoters that are capable of expressing a DNA molecule inplant plastids are a psbA promoter or a rbcL promoter. A plastidexpression cassette also preferably further comprises a plastid gene 3′untranslated sequence (3′UTR) operatively linked to a DNA molecule ofthe present invention. The role of untranslated sequences is preferablyto direct the 3′ processing of the transcribed RNA rather thantermination of transcription. Preferably, the 3′ UTR is a plastid rps16gene 3′ untranslated sequence or the Arabidopsis plastid psbA gene 3′untranslated sequence. In a further preferred embodiment, a plastidexpression cassette comprises a poly-G tract instead of a 3′untranslated sequence. A plastid expression cassette also preferablyfurther comprises a 5′ untranslated sequence (5′UTR) functional in plantplastids operatively linked to a DNA molecule of the present invention.

[0140] A plastid expression cassette is comprised in a plastidtransformation vector, which preferably further comprises flankingregions for integration into the plastid genome by homologousrecombination. The plastid transformation vector may optionally compriseat least one plastid origin of replication. The present invention alsoencompasses a plant plastid transformed with such a plastidtransformation vector, wherein the DNA molecule is expressible in theplant plastid. The invention also encompasses a plant or plant cell,including the progeny thereof, comprising this plant plastid. In apreferred embodiment, the plant or plant cell, including the progenythereof, is homoplasmic for transgenic plastids.

[0141] Other promoters that are capable of expressing a DNA molecule inplant plastids are transactivator-regulated promoters, preferablyheterologous with respect to the plant or to the subcellular organelleor component of the plant cell in which expression is effected. In thesecases, the DNA molecule encoding the transactivator is inserted into anappropriate nuclear expression cassette which is transformed into theplant nuclear DNA. The transactivator is targeted to plastids using aplastid transit peptide. The transactivator and thetransactivator-driven DNA molecule are brought together either bycrossing to a selected plastid-transformed line a transgenic linecontaining a DNA molecule encoding the transactivator supplemented witha plastid-targeting sequence and operably linked to a nuclear promoter,or by directly transforming a plastid transformation vector containingthe desired DNA molecule into a transgenic line containing a DNAmolecule encoding the transactivator supplemented with aplastid-targeting sequence and operably linked to a nuclear promoter. Ifthe nuclear promoter is an inducible promoter, in particular achemically inducible promoter, expression of the DNA molecule in theplastids of plants is activated by foliar application of a chemicalinducer. Such inducible transactivator-mediated plastid expressionsystem is preferably tightly regulatable, with no detectable expressionprior to induction and exceptionally high expression and accumulation ofprotein following induction. A preferred transactivator is for exampleviral RNA polymerase. Preferred promoters of this type are promotersrecognized by a single sub-unit RNA polymerase, such as the T7 gene 10promoter, which is recognized by the bacteriophage T7 DNA-dependent RNApolymerase. The gene encoding the T7 polymerase is preferablytransformed into the nuclear genome and the T7 polymerase is targeted tothe plastids using a plastid transit peptide. Promoters suitable fornuclear expression of a gene, for example a gene encoding a viral RNApolymerase such as the T7 polymerase, are described infra or supra.Expression of the DNA molecules in plastids can be constitutive or canbe inducible Expression of the DNA molecules in the plastids can be alsoorgan- or tissue-specific. These different embodiment are extensivelydescribed in WO 98/11235, incorporated herein by reference. Thus, in oneaspect, the present invention has coupled expression in the nucleargenome of a choroplast-targeted phage T7 RNA polymerase under control ofthe chemically inducible PR-1a promoter (U.S. Pat. No. 5,614,395incorporated by reference) of tobacco to a chloroplast reportertransgene regulated by T7 gene 10 promoter/terminator sequences. Forexample, when plastid transformants homoplasmic for the maternallyinherited trehalose biosynthetis genes are pollinated by linesexpressing the T7 polymerase in the nucleus, F1 plants are obtained thatcarry both transgene constructs but do not express them. Synthesis oflarge amounts of enzymatically active protein is triggered in plastidsof these plants only after foliar application of the PR-1a inducercompound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester(BTH).

[0142] In a preferred embodiment, two or more genes, e.g. trehalosebiosynthetic genes, are transcribed from the plastid genome from asingle promoter in an operon-like polycistronic gene. In a preferredembodiment, the operon-like polycistronic gene comprises an interveningDNA sequence between two genes in the operon-like polycistronic gene. Ina preferred embodiment, the DNA sequence is not present in the plastidgenome to avoid homologous recombination with plastid sequences. Inanother preferred embodiment, the DNA sequence is derived from the 5′untranslated (UTR) region of a non-eukaryotic gene, preferably from aviral 5′UTR, preferably from a 5′UTR derived from a bacterial phage,such as a T7, T3 or SP6 phage. In a preferred embodiment, the DNAsequence is modified to prevent the formation of RNA secondarystructures in a RNA transcript of the operon-like polycistronic gene,e.g. between the DNA sequence and the rbs of the downstream gene. Suchsecondary structures would inhibit or repress the expression of thedownstream gene, particularly the initiation of its translation. SuchRNA secondary structures are predicted by determining their meltingtemperatures using computer models and programs such a the “mfold”program version 3 (by Zuker and Turner, Washington University School ofMedicine, St-Louis, Mo.) and other methods well known to one skilled inthe art. Such a DNA sequence is exemplified below.

[0143] The presence of the intervening DNA sequence in the operon-likepolycistronic gene increases the accessibility of the rbs of thedownstream gene, thus resulting in higher rates of expression. Suchstrategy is applicable to any two or more genes to be transcribed fromthe plastid genome from a single promoter in an operon-like chimericgene. Such genes can be part of a metabolic pathway, or are genesencoding input or output traits. Example of metabolic pathways are e.g.sugar biosynthetic pathways, such as trehalose or fructans.

[0144] In a further embodiment, the DNA molecules of the presentinvention are modified by incorporation of random mutations in atechnique known as in-vitro recombination or DNA shuffling. Thistechnique is described in Stemmer et al., Nature 370: 389-391 (1994) andU.S. Pat. No. 5,605,793 incorporated herein by reference. Millions ofmutant copies of the nucleotide sequences are produced based on theoriginal nucleotide sequence described herein and variants with improvedproperties, such as increased activity or altered specificity arerecovered. The method encompasses forming a mutagenized double-strandedpolynucleotide from a template double-stranded polynucleotide comprisingthe nucleotide sequence of this invention, wherein the templatedouble-stranded polynucleotide has been cleaved intodouble-stranded-random fragments of a desired size, and comprises thesteps of adding to the resultant population of double-stranded randomfragments one or more single or double-stranded oligonucleotides,wherein said oligonucleotides comprise an area of identity and an areaof heterology to the double-stranded template polynucleotide; denaturingthe resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; incubating theresultant population of single-stranded fragments with a polymeraseunder conditions which result in the annealing of said single-strandedfragments at said areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprime replication of the other, thereby forming a mutagenizeddoubles-tranded polynucleotide; and repeating the second and third stepsfor at least two further cycles, wherein the resultant mixture in thesecond step of a further cycle includes the mutagenized double-strandedpolynucleotide from the third step of the previous cycle, and thefurther cycle forms a further mutagenized double-strandedpolynucleotide. In a preferred embodiment, the concentration of a singlespecies of double-stranded random fragment in the population ofdouble-stranded random fragments is less than 1% by weight of the totalDNA. In a further preferred embodiment, the template double-strandedpolynucleotide comprises at least about 100 species of polynucleotides.In another embodiment, the size of the double-stranded random fragmentsis from about 5 bp to 5 kb. In a further embodiment, the fourth step ofthe method comprises repeating the second and the third steps for atleast 10 cycles.

[0145] Numerous transformation vectors are available for planttransformation, and the genes of this invention can be used inconjunction with any such vectors. The selection of vector for use willdepend upon the preferred transformation technique and the targetspecies for transformation. For certain target species, differentantibiotic or herbicide selection markers may be preferred. Selectionmarkers used routinely in transformation include the nptll gene whichconfers resistance to kanamycin and related antibiotics (Messing &Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187(1983)), the bar gene which confers resistance to the herbicidephosphinothricin (White et al., Nucl! Acids Res 18: 1062 (1990), Spenceret al. Theor Appl Genet 79: 625-631(1990)), the hpt gene which confersresistance to the antibiotic hygromycin (Blochinger & Diggelmann, MolCell Biol 4: 2929-2931), and the dhfr gene, which confers resistance tomethatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)).

[0146] Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) andpXYZ. Below the construction of two typical vectors is described.

[0147] Construction of PCIB200 and pCIB2001: The binary vectors pCIB200and pCIB2001 are used for the construction of recombinant vectors foruse with Agrobacterium and was constructed in the following manner.pTJS75kan was created by Narl digestion of pTJS75 (Schmidhauser &Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of thetetracycline-resistance gene, followed by insertion of an Accl fragmentfrom pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982);Bevan et al., Nature 304: 184-187 (1983); McBride et al., PlantMolecular Biology 14: 266-276 (1990)). XhoI linkers were ligated to theEcoRV fragment of pCIB7 which contains the left and right T-DNA borders,a plant selectable nos/nptII chimeric gene and the pUC polylinker(Rothstein et al., Gene 53: 153-161 (1987)), and the XhoI-digestedfragment was cloned into SaII-digested pTJS75kan to create pCIB200 (seealso EP 0 332 104, example 19). pCIB200 contains the following uniquepolylinker restriction sites: EcoRI, SstI, KpnI, BgIII, XbaI, and SaII.pCIB2001 is a derivative of pCIB200 which created by the insertion intothe polylinker of additional restriction sites. Unique restriction sitesin the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BgIII, XbaI, SaII,MIuI, BcII, AvrII, ApaI, HpaI, and StuI. pCIB2001, in addition tocontaining these unique restriction sites also has plant and bacterialkanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

[0148] Construction of pCIB10 and Hygromycin Selection Derivativesthereof: The binary vector pCIB10 contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al. (Gene 53: 153-161 (1987)).Various derivatives of pCIB10 have been constructed which incorporatethe gene for hygromycin B phosphotransferase described by Gritz et al.(Gene 25: 179-188 (1983)). These derivatives enable selection oftransgenic plant cells on hygromycin only (pCIB743), or hygromycin andkanamycin (pCIB715, pCIB717).

[0149] Transformation without the use of Agrobacterium tumefacienscircumvents the requirement for T-DNA sequences in the chosentransformation vector and consequently vectors lacking these sequencescan be utilized in addition to vectors such as the ones described abovewhich contain T-DNA sequences. Transformation techniques which do notrely on Agrobacterium include transformation via particle bombardment,protoplast uptake (e.g. PEG and electroporation) and microinjection. Thechoice of vector depends largely on the preferred selection for thespecies being transformed. Below, the construction of some typicalvectors is described.

[0150] Construction of pCIB3064: pCIB3064 is a pUC-derived vectorsuitable for direct gene transfer techniques in combination withselection by the herbicide basta (or phosphinothricin). The plasmidpCIB246 comprises the CaMV 35S promoter in operational fusion to the E.coli GUS gene and the CaMV 35S transcriptional terminator and isdescribed in the PCT published application WO 93/07278. The 35S promoterof this vector contains two ATG sequences 5′ of the start site. Thesesites were mutated using standard PCR techniques in such a way as toremove the ATGs and generate the restriction sites SspI and PvuII. Thenew restriction sites were 96 and 37 bp away from the unique Sall siteand 101 and 42 bp away from the actual start site. The resultantderivative of pCIB246 was designated pCIB3025. The GUS gene was thenexcised from pCIB3025 by digestion with Sall and SacI, the terminirendered blunt and religated to generate plasmid pCIB3060. The plasmidpJIT82 was obtained from the John Innes Centre, Norwich and the a 400 bpSmal fragment containing the bar gene from Streptomycesviridochromogenes was excised and inserted into the HpaI site ofpCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generatedpCIB3064 which comprises the bar gene under the control of the CaMV 35Spromoter and terminator for herbicide selection, a gene fro ampicillinresistance (for selection in E. coli) and a polylinker with the uniquesites SphI, PstI, HindIII, and BamHI. This vector is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

[0151] Construction of pSOG19 and pSOG35: pSOG35 is a transformationvector which utilizes the E. coli gene dihydrofolate reductase (DHFR) asa selectable marker conferring resistance to methotrexate. PCR was usedto amplify the 35S promoter (˜800 bp), intron 6 from the maize AdH1 gene(˜550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10.A 250 bp fragment encoding the E. coli dihydrofolate reductase type IIgene was also amplified by PCR and these two PCR fragments wereassembled with a SacI-PstI fragment from pbI221 (Clontech) whichcomprised the pUC19 vector backbone and the nopaline synthaseterminator. Assembly of these fragments generated pSOG19 which containsthe 35S promoter in fusion with the intron 6 sequence, the GUS leader,the DHFR gene and the nopaline synthase terminator. Replacement of theGUS leader in pSOG19 with the leader sequence from Maize ChloroticMottle Virus (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carrythe pUC gene for ampicillin resistance and have HindIII, SphI, PstI andEcoRI sites available for the cloning of foreign sequences.

[0152] Gene sequences intended for expression in transgenic plants arefirstly assembled in expression cassettes behind a suitable promoter andupstream of a suitable transcription terminator. These expressioncassettes can then be easily transferred to the plant transformationvectors described above.

[0153] The selection of promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and this selection will reflect the desiredlocation of biosynthesis of the enzyme. Alternatively, the selectedpromoter may drive expression of the gene under a light-induced or othertemporally regulated promoter. A further (and preferred) alternative isthat the selected promoter be inducible by an external stimulus, e.g.,application of a specific chemical inducer or wounding. This wouldprovide the possibility of inducing trehalose biosynthetic genetranscription only when desired.

[0154] A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators and those which are known tofunction in plants and include the CaMV 35S terminator, the tmlterminator, the nopaline synthase terminator, the pea rbcS E9terminator. These can be used in both monocotyledons and dicotyledons.

[0155] Numerous sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

[0156] Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adh1 gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develep 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

[0157] A number of non-translated leader sequences derived from virusesare also known to enhance expression, and these are particularlyeffective in dicotyledonous cells. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “Ω-sequence“), Maize Chlorotic MottleVirus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res.15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15; 65-79(1990)).

[0158] Various mechanisms for targeting gene products are known to existin plants and the sequences controlling the functioning of thesemechanisms have been characterized in some detail. For example, thetargeting of gene products to the chloroplast is controlled by a signalsequence found at the aminoterminal end of various proteins and which iscleaved during chloroplast import yielding the mature protein (e.g.Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signalsequences can be fused to heterologous gene products to effect theimport of heterologous products into the chloroplast (van den Broeck etal. Nature 313: 358-363 (1985)). DNA encoding for appropriate signalsequences can be isolated from the 5′ end of the cDNAs encoding theRUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2protein and many other proteins which are known to be chloroplastlocalized.

[0159] Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting to cellular protein bodies has been described byRogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

[0160] In addition, sequences have been characterized which cause thetargeting of gene products to other cell compartments. Aminoterminalsequences are responsible for targeting to the ER, the apoplast, andextracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). Additionally, aminoterminal sequences in conjunctionwith carboxyterminal sequences are responsible for vacuolar targeting ofgene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

[0161] By the fusion of the appropriate targeting sequences describedabove to transgene sequences of interest it is possible to direct thetransgene product to any organelle or cell compartment. For chloroplasttargeting, for example, the chloroplast signal sequence from the RUBISCOgene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused inframe to the aminoterminal ATG of the transgene. The signal sequenceselected should include the known cleavage site and the fusionconstructed should take into account any amino acids after the cleavagesite which are required for cleavage. In some cases this requirement maybe fulfilled by the addition of a small number of amino acids betweenthe cleavage site and the transgene ATG or alternatively replacement ofsome amino acids within the transgene sequence. Fusions constructed forchloroplast import can be tested for efficacy of chloroplast uptake byin vitro translation of in vitro transcribed constructions followed byin vitro chloroplast uptake using techniques described by (Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier. pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205:446-453 (1986)). These construction techniques are well known in the artand are equally applicable to mitochondria and peroxisomes. The choiceof targeting which may be required for trehalose biosynthetic genes willdepend on the cellular localization of the precursor required as thestarting point for a given pathway. This will usually be cytosolic orchloroplastic, although it may is some cases be mitochondrial orperoxisomal.

[0162] The above-described mechanisms for cellular targeting can beutilized not only in conjunction with their cognate promoters, but alsoin conjunction with heterologous promoters so as to effect a specificcell targeting goal under the transcriptional regulation of a promoterwhich has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

[0163] The present invention encompasses the expression of trehalosebiosynthetic genes under the regulation of any promoter that isexpressible in plants, regardless of the origin of the promoter.

[0164] Furthermore, the invention encompasses the use of anyplant-expressible promoter in conjunction with any further sequencesrequired or selected for the expression of the trehalose biosyntheticgene. Such sequences include, but are not restricted to, transcriptionalterminators, extraneous sequences to enhance expression (such as introns[e.g. Adh intron 1], viral sequences [e. g. TMV-Ω]), and sequencesintended for the targeting of the gene product to specific organellesand cell compartments.

[0165] Suitable plant-expressible promoters are those that are expressedconstitutively such as the CaMV 35S promoter, the actin promoter or theubiquitin promoter.

[0166] Construction of the plasmid pCGN1761 containing the “double” 35Spromoter is described in the published patent application EP 0 392 225(example 23). pCGN1761 contains the “double” 35S promoter and the tmltranscriptional terminator with a unique EcoRI site between the promoterand the terminator and has a pUC-type backbone. A derivative of pCGN1761was constructed which has a modified polylinker which includes Notl andXhoI sites in addition to the existing EcoRI site. This derivative wasdesignated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNAsequences or gene sequences (including microbial ORF sequences) withinits polylinker for the purposes of their expression under the control ofthe 35S promoter in transgenic plants. The entire 35S promoter-genesequence-tmI terminator cassette of such a construction can be excisedby HindIII, SphI, SaII, and Xbal sites 5′ to the promoter and XbaI,BamHI and BgII sites 3′ to the terminator for transfer to transformationvectors such as those described above. Furthermore, the double 35Spromoter fragment can be removed by 5′ excision with HindIII, SphI,SaII, XbaI, or PstI, and 3′ excision with any of the polylinkerrestriction sites (EcoRI, Notl or XhoI) for replacement with anotherpromoter.

[0167] For any of the constructions described in this section,modifications around the cloning sites can be made by the introductionof sequences which may enhance translation. This is particularly usefulwhen genes derived from microorganisms are to be introduced into plantexpression cassettes as these genes may not contain sequences adjacentto their initiating methionine which may be suitable for the initiationof translation in plants. In cases where genes derived frommicroorganisms are to be cloned into plant expression cassettes at theirATG it may be useful to modify the site of their insertion to optimizetheir expression. Modification of pCGN1761 ENX by optimization of thetranslational initiation site is described by way of example toincorporate one of several optimized sequences for plant expression(e.g. Joshi, supra).

[0168] Further plant-expressible promoters that can be suitably usedwithin the scope of the present invention are chemically regulatablepromoters such as those described hereinafter. For example, this sectiondescribes the replacement of the double 35S promoter in pCGN1761ENX withany promoter of choice; by way of example, the chemically regulatablePR-1a promoter is described in U.S. Pat. No. 5,614,395, which is herebyincorporated by reference in its entirety, and the chemicallyregulatable Arabidopsis PR-1 promoter is described in U.S. ProvisionalApplication No. 60/027,228, incorporated herein by reference. Thepromoter of choice is preferably excised from its source by restrictionenzymes, but can alternatively be PCR-amplified using primers whichcarry appropriate terminal restriction sites. Should PCR-amplificationbe undertaken, then the promoter should be resequenced to check foramplification errors after the cloning of the amplified promoter in thetarget vector. The chemically regulatable tobacco PR-1a promoter iscleaved from plasmid pCIB1004 (see EP 0 332 104, example 21 forconstruction) and transferred to plasmid pCGN1761ENX. pCIB1004 iscleaved with Ncol and the resultant 3′ overhang of the linearizedfragment is rendered blunt by treatment with T4 DNA polymerase. Thefragment is then cleaved with HindIII and the resultant PR-1a promotercontaining fragment is gel purified and cloned into pCGN1761ENX fromwhich the double 35S promoter has been removed. This is done by cleavagewith XhoI and blunting with T4 polymerase, followed by cleavage withHindIII and isolation of the larger vector-terminator containingfragment into which the pCIB1004 promoter fragment is cloned. Thisgenerates a pCGN1761ENX derivative with the PR-1a promoter and the tmIterminator and an intervening polylinker with unique EcoRI and Notlsites. Selected trehalose biosynthetic genes can be inserted into thisvector, and the fusion products (i.e. promoter-gene-terminator) cansubsequently be transferred to any selected transformation vector,including those described in this application.

[0169] Various chemical regulators may be employed to induce expressionof the trehalose biosynthetic coding sequence in the plants transformedaccording to the present invention. In the context of the instantdisclosure, “chemical regulators” include chemicals known to be inducersfor the PR-1a promoter in plants, or close derivatives thereof. Apreferred group of regulators for the chemically inducible trehalosebiosynthetic genes of this invention is based on thebenzo-1,2,3-thiadiazole (BTH) structure and includes, but is not limitedto, the following types of compounds: benzo-1,2,3-thiadiazolecarboxylicacid, benzo-1,2,3-thiadiazolethiocarboxylic acid,cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylic acidamide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide,benzo-1,2,3-thiadiazole-7-carboxylic acid,benzo-1,2,3-thiadiazole-7-thiocarboxylic acid,7-cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazole-7-carboxylicacid amide, benzo-1,2,3-thiadiazole-7-carboxylic acid hydrazide, alkylbenzo-1,2,3-thiadiazolecarboxylate in which the alkyl group contains oneto six carbon atoms, methyl benzo-1,2,3-thiadiazole-7-carboxylate,n-propyl benzo-1,2,3-thiadiazole-7carboxylate, benzylbenzo-1,2,3-thiadiazole-7-carboxylate,benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide, andsuitable derivatives thereof. Other chemical inducers may include, forexample, benzoic acid, salicylic acid (SA), polyacrylic acid andsubstituted derivatives thereof; suitable substituents include loweralkyl, lower alkoxy, lower alkylthio, and halogen. Still another groupof regulators for the chemically inducible DNA sequences of thisinvention is based on the pyridine carboxylic acid structure, such asthe isonicotinic acid structure and preferably the haloisonicotinic acidstructure. Preferred are dichloroisonicotinic acids and derivativesthereof, for example the lower alkyl esters. Suitable regulators of thisclass of compounds are, for example, 2,6-dichloroisonicotinic acid(INA), and the lower alkyl esters thereof, especially the methyl ester.

[0170] Constitutive Expression can also be achieved by the ActinPromoter. Several isoforms of actin are known to be expressed in mostcell types and consequently the actin promoter is a good choice for aconstitutive promoter. In particular, the promoter from the rice Act1gene has been cloned and characterized (McElroy et al. Plant Cell 2:163-171 (1990)). A 1.3 kb fragment of the promoter was found to containall the regulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the Act1promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the Act1-intron1, AdH1 5′ flanking sequence and AdH1-intron 1 (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and the Act1 intron orthe Act1 5′ flanking sequence and the Act1 intron. Optimization ofsequences around the initiating ATG (of the GUS reporter gene) alsoenhanced expression. The promoter expression cassettes described byMcElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easilymodified for the expression of trehalose biosynthetic genes and areparticularly suitable for use in monocotyledonous hosts. For example,promoter containing fragments can be removed from the McElroyconstructions and used to replace the double 35S promoter inpCGN1761ENX, which is then available for the insertion or specific genesequences. The fusion genes thus constructed can then be transferred toappropriate transformation vectors. In a separate report the rice Act1promoter with its first intron has also been found to direct highexpression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12:506-509 (1993)).

[0171] Ubiquitin is another gene product known to accumulate in manycell types and its promoter has been cloned from several species for usein transgenic plants (e.g. sunflower—Binet et al. Plant Science 79:87-94 (1991), maize—Christensen et al. Plant Molec. Biol. 12: 619-632(1989)) for constitutive expression. The maize ubiquitin promoter hasbeen developed in transgenic monocot systems and its sequence andvectors constructed for monocot transformation are disclosed in thepatent publication EP 0 342 926 (to Lubrizol). Further, Taylor et al.(Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) whichcomprises the maize ubiquitin promoter and first intron and its highactivity in cell suspensions of numerous monocotyledons when introducedvia microprojectile bombardment. The ubiquitin promoter is suitable forthe expression of trehalose biosynthetic genes in transgenic plants,especially monocotyledons. Suitable vectors are derivatives of pAHC25 orany of the transformation vectors described in this application,modified by the introduction of the appropriate ubiquitin promoterand/or intron sequences.

[0172] Another pattern of expression for the enzymes of the instantinvention is root expression. A suitable root promoter is that describedby de Framond (FEBS 290: 103-106 (1991)) and also in the publishedpatent application EP 0 452 269 (to Ciba-Geigy). This promoter istransferred to a suitable vector such as pCGN1761ENX for the insertionof a trehalose biosynthetic gene and subsequent transfer of the entirepromoter-gene-terminator cassette to a transformation vector ofinterest.

[0173] Wound-inducible promoters may also be suitable for the expressionof trehalose biosynthetic genes. Numerous such promoters have beendescribed (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993),Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, PlantMolec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and all aresuitable for use with the instant invention. Logemann et al. describethe 5′ upstream sequences of the dicotyledonous potato wun1 gene. Xu etal. show that a wound inducible promoter from the dicotyledon potato(pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehledescribe the cloning of the maize Wip1 cDNA which is wound induced andwhich can be used to isolated the cognate promoter using standardtechniques. Similarly, Firek et al. and Warner et al. have described awound induced gene from the monocotyledon Asparagus officinalis which isexpressed at local wound and pathogen invasion sites. Using cloningtechniques well known in the art, these promoters can be transferred tosuitable vectors, fused to the trehalose biosynthetic genes of thisinvention, and used to express these genes at the sites of plantwounding.

[0174] Patent Application WO 93/07278 (to Ciba-Geigy) describes theisolation of the maize trpA gene which is preferentially expressed inpith cells. The gene sequence and promoter extend up to -1726 from thestart of transcription are presented. Using standard molecularbiological techniques, this promoter or parts thereof, can betransferred to a vector such as pCGN1761 where it can replace the 35Spromoter and be used to drive the expression of a foreign gene in apith-preferred manner. In fact, fragments containing the pith-preferredpromoter or parts thereof can be transferred to any vector and modifiedfor utility in transgenic plants.

[0175] A maize gene encoding phosphoenol carboxylase (PEPC) has beendescribed by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)).Using standard molecular biological techniques the promoter for thisgene can be used to drive the expression of any gene in a leaf-specificmanner in transgenic plants.

[0176] Chen & Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) havedescribed the successful use of a chloroplast transit peptide for importof a heterologous transgene. This peptide used is the transit peptidefrom the rbcS gene from Nicotiana plumbaginifolia (Poulsen et al. Mol.Gen. Genet. 205: 193-200 (1986)). Using the restriction enzymes DraI andSphI, or Tsp509I and SphI the DNA sequence encoding this transit peptidecan be excised from plasmid prbcS-8B and manipulated for use with any ofthe constructions described above. The DraI-SphI fragment extends from−58 relative to the initiating rbcS ATG to, and including, the firstamino acid (also a methionine) of the mature peptide immediately afterthe import cleavage site, whereas the Tsp509I-SphI fragment extends from-8 relative to the initiating rbcS ATG to, and including, the firstamino acid of the mature peptide. Thus, these fragments can beappropriately inserted into the polylinker of any chosen expressioncassette generating a transcriptional fusion to the untranslated leaderof the chosen promoter (e.g. 35S, PR-1a, actin, ubiquitin etc.), whilstenabling the insertion of a trehalose biosynthetic gene in correctfusion downstream of the transit peptide. Constructions of this kind areroutine in the art. For example, whereas the DraI end is already blunt,the 5′ Tsp509I site may be rendered blunt by T4 polymerase treatment, ormay alternatively be ligated to a linker or adaptor sequence tofacilitate its fusion to the chosen promoter. The 3′ SphI site may bemaintained as such, or may alternatively be ligated to adaptor of linkersequences to facilitate its insertion into the chosen vector in such away as to make available appropriate restriction sites for thesubsequent insertion of a selected trehalose biosynthetic gene. Ideallythe ATG of the SphI site is maintained and comprises the first ATG ofthe selected trehalose biosynthetic gene. Chen & Jagendorf provideconsensus sequences for ideal cleavage for chloroplast import, and ineach case a methionine is preferred at the first position of the matureprotein. At subsequent positions there is more variation and the aminoacid may not be so critical. In any case, fusion constructions can beassessed for efficiency of import in vitro using the methods describedby Bartlett et al. (In: Edelmann et al. (Eds.) Methods in ChloroplastMolecular Biology, Elsevier. pp 1081-1091 (1982)) and Wasmann et al.(Mol. Gen. Genet. 205: 446453 (1986)). Typically the best approach maybe to generate fusions using the selected trehalose biosynthetic genewith no modifications at the aminoterminus, and only to incorporatemodifications when it is apparent that such fusions are not chloroplastimported at high efficiency, in which case modifications may be made inaccordance with the established literature (Chen & Jagendorf; Wasman etal.; Ko & Ko, J. Biol. Chem. 267: 13910-13916 (1992)).

[0177] A preferred vector is constructed by transferring the DraI-SphItransit peptide encoding fragment from prbcS-8B to the cloning vectorpCGN1761ENX/Sph-. This plasmid is cleaved with EcoRI and the terminirendered blunt by treatment with T4 DNA polymerase. Plasmid prbcS-8B iscleaved with SphI and ligated to an annealed molecular adaptor. Theresultant product is 5′-terminally phosphorylated by treatment with T4kinase. Subsequent cleavage with DraI releases the transit peptideencoding fragment which is ligated into the blunt-end ex-EcoRI sites ofthe modified vector described above. Clones oriented with the 5′ end ofthe insert adjacent to the 3′ end of the 35S promoter are identified bysequencing. These clones carry a DNA fusion of the 35S leader sequenceto the rbcS-8A promoter-transit peptide sequence extending from -58relative to the rbcS ATG to the ATG of the mature protein, and includingat that position a unique SphI site, and a newly created EcoRI site, aswell as the existing NotI and XhoI sites of pCGN1761ENX. This new vectoris designated pCGN1761/CT. DNA sequences are transferred to pCGN1761/CTin frame by amplification using PCR techniques and incorporation of anSphI, NSphI, or NIaIII site at the amplified ATG, which followingrestriction enzyme cleavage with the appropriate enzyme is ligated intoSphI-cleaved pCGN1761/CT. To facilitate construction, it may be requiredto change the second amino acid of cloned gene, however, in almost allcases the use of PCR together with standard site directed mutagenesiswill enable the construction of any desired sequence around the cleavagesite and first methionine of the mature protein.

[0178] A further preferred vector is constructed by replacing the double35S promoter of pCGN1761 ENX with the BamHI-SphI fragment of prbcS-8Awhich contains the full-length light regulated rbcS-8A promoter from-1038 (relative to the transcriptional start site) up to the firstmethionine of the mature protein. The modified pCGN1761 with thedestroyed SphI site is cleaved with PstI and EcoRI and treated with T4DNA polymerase to render termini blunt. prbcS-8A is cleaved SphI andligated to the annealed molecular adaptor of the sequence describedabove. The resultant product is 5′-terminally phosphorylated bytreatment with T4 kinase. Subsequent cleavage with BamHI releases thepromoter-transit peptide containing fragment which is treated with T4DNA polymerase to render the BamHI terminus blunt. The promoter-transitpeptide fragment thus generated is cloned into the prepared pCGN1761ENXvector, generating a construction comprising the rbcS-8A promoter andtransit peptide with an SphI site located at the cleavage site forinsertion of heterologous genes. Further, downstream of the SphI sitethere are EcoRI (re-created), NotI, and XhoI cloning sites. Thisconstruction is designated pCGN1761rbcS/CT.

[0179] Similar manipulations can be undertaken to utilize other GS2chloroplast transit peptide encoding sequences from other sources(monocotyledonous and dicotyledonous) and from other genes. In addition,similar procedures can be followed to achieve targeting to othersubcellular compartments such as mitochondria.

[0180] Transformation techniques for dicotyledons are well known in theart and include Agrobacterium-based techniques and techniques which donot require Agrobacterium. NonAgrobacterium techniques involve theuptake of exogenous genetic material directly by protoplasts or cells.This can be accomplished by PEG or electroporation mediated uptake,particle bombardment-mediated delivery, or microinjection. Examples ofthese techniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

[0181] Agrobacterium-mediated transformation is a preferred techniquefor transformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species. Themany crop species which are routinely transformable by Agrobacteriuminclude tobacco, tomato, sunflower, cotton, oilseed rape, potato,soybean, alfalfa and poplar (EP 0 317 511 (cotton [1313]), EP 0 249 432(tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), U.S. Pat. No.4,795,855 (poplar)). Agrobacterium transformation typically involves thetransfer of the binary vector carrying the foreign DNA of interest (e.g.pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which maydepend of the complement of vir genes carried by the host Agrobacteriumstrain either on a co-resident Ti plasmid or chromosomally (e.g. strainCIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169(1993)). The transfer of the recombinant binary vector to Agrobacteriumis accomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877(1988)).

[0182] Transformation of the target plant species by recombinantAgrobacterium usually involves co-cultivation of the Agrobacterium withexplants from the plant and follows protocols well known in the art.Transformed tissue is regenerated on selectable medium carrying theantibiotic or herbicide resistance marker present between the binaryplasmid T-DNA borders.

[0183] Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complex vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

[0184] Patent Applications EP 0 292 435 ([1280/1281] to Ciba-Geigy), EP0 392 225 (to Ciba-Geigy) and WO 93/07278 (to Ciba-Geigy) describetechniques for the preparation of callus and protoplasts from an 6Iiteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Frommet al. (Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al.(Biotechnology 11: 194-200 (1993)) describe techniques for thetransformation of élite inbred lines of maize by particle bombardment.This technique utilizes immature maize embryos of 1.5-2.5 mm lengthexcised from a maize ear 14-15 days after pollination and a PDS-1000HeBiolistics device for bombardment.

[0185] Transformation of rice can also be undertaken by direct genetransfer techniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

[0186] Patent Application EP 0 332 581 (to Ciba-Geigy) describestechniques for the generation, transformation and regeneration ofPooideae protoplasts. These techniques allow the transformation ofDactylis and wheat. Furthermore, wheat transformation was been describedby Vasil et al. (Biotechnology 10: 667-674 (1992)) using particlebombardment into cells of type C long-term regenerable callus, and alsoby Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al.(Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment ofimmature embryos and immature embryo-derived callus. A preferredtechnique for wheat transformation, however, involves the transformationof wheat by particle bombardment of immature embryos and includes eithera high sucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos whichis allowed to proceed in the dark. On the chosen day of bombardment,embryos are removed from the induction medium and placed onto theosmoticum (i.e. induction medium with sucrose or maltose added at thedesired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 h and are then bombarded. Twenty embryos per targetplate is typical, although not critical. An appropriate gene-carryingplasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer sizegold particles using standard procedures. Each plate of embryos is shotwith the DuPont Biolistics® helium device using a burst pressure of˜1000 psi using a standard 80 mesh screen. After bombardment, theembryos are placed back into the dark to recover for about 24 h (stillon osmoticum). After 24 hrs, the embryos are removed from the osmoticumand placed back onto induction medium where they stay for about a monthbefore regeneration. Approximately one month later the embryo explantswith developing embryogenic callus are transferred to regenerationmedium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing theappropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2mg/l methotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” which contained half-strength MS, 2% sucrose, and the sameconcentration of selection agent. Patent application 08/147,161describes methods for wheat transformation and is hereby incorporated byreference.

[0187] The invention will be further described by reference to thefollowing detailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLES

[0188] A. Expression of the trehalose-6-phosphate synthase andtrehalose-6-phosphate phosphatase genes in the plant cytosol

Example 1

[0189] Preparation of a Chimeric Gene Containing the E. coliTrehalose-6-phosphate Synthase Gene Fused to the Tobacco PR-1a Promoter

[0190] Plasmid pCGN4467 containing the coding sequence of the E colitrehalose-6-phosphate synthase gene (OtsA, Kaasen et al. (1994) Gene 145(1), 9-15, EMBL/UGenbank accession number X69160) under the control of adouble 35S promoter and fused to the tml3′ polyadenylation signals(pCGN4467 is a derivative of pCGN1761, EP 0392225) is used as templatefor PCR with a left-to-right “topstrand” primer including the ATGpreceded by a GCC codon and followed by a newly added GCA codon, thuscreating a Ncol restriction site at the ATG, and the first 24 bases ofthe OtsA gene (primer TREA+: GTC AGC CAT GGC MG TCG TTT AGT CGT AGT ATCTAA C, SEQ ID No:1) and a right-to-left “bottomstrand” primer homologousto positions 392 to 416 downstream of the new ATG (primer TREA-: GCA MTGGC MC AGG TGA TAA TCG, SEQ ID No:2). This PCR reaction is undertakenwith AmpliTaq DNA polymerase according to the manufacturer'srecommendations (Perkin Elmer/Roche, Branchburg, N.J.) for five cyclesat 94° C. (30 s), 40° C. (60 s), and 72° C. (30 s) followed by 25 cyclesat 94° C. (30 s), 55° C. (60 s) and 72°C. (30 s) and this generated aproduct of 423 bp containing a NcoI site at its left end and a BamHIsite at its right end. The fragment is gel purified using standardprocedures, cleaved with NcoI and BamHI (all restriction enzymes arepurchased from Promega, Madison, Wis.) and ligated into the NcoI andBamHI sites of pUC21 which is a pUC derivative containing a polylinkerwith the following restriction sites: SpeI/Stul/XhoI/BgIII/CIAI/Nsil/SphI/NcoI/KpnI/XmaI/SmaI/SacI/EcoRI/BstIBI/HindIII/PstI/MluI/SaII/AatII/NdeI/BamHI/EcoRV/NotI/EagI/XbaI/SpeIto obtain pUCOTSA.

[0191] Plasmid pUCOTSA is then digested with SpeI and BamHI, the 400bpfragment containing the 5′ end of the OtsA gene is gel purified andligated with pCGN4467 that had previously been digested with XbaI andBamHI, to obtain pCGNOTSA containing the entire OtsA gene. PlasmidpCGNOTSA is digested with NcoI and SacI, the 1.4 kb long fragmentcontaining the OtsA gene is gel purified and ligated into the NcoI andSacI sites of pJG203 between a 903 bp long tobacco PR-1a promoter andthe nos gene termination signals (Uknes et al. (1993), The Plant Cell5,159-169). Plasmid pJG203 is a derivative of pBSGusl.2 (Uknes et al.(1993), The Plant Cell 5,159-169), comprising a 903 bp long tobaccoPR-1a promoter fused to the GUS gene and nos polyadenylation signals. InpJG203, the second SacI site at the end of the nos polyadenylationsignals has been removed by partial digestion with SacI, filling-in ofthe protruding ends and religation. Plasmid pPR10TSA containing the OtsAgene fused to the tobacco PR-1a promoter is thus obtained.

Example 2

[0192] Preparation of a Chimeric Gene Containing the E. coliTrehalose-6-phosphate Phosphatase Gene Fused to the Tobacco PR-1aPromoter

[0193] Plasmid pCGN4452 containing the coding sequence of the E. colitrehalose-6-phosphate phosphatase gene (OtsB, Kaasen et al. (1994) Gene145 (1), 9-15, EMBUGenbank accession number X69160) under the control ofa double 35S promoter and fused to the tml3′ polyadenylation signals(pCGN4452 is a derivative of pCGN1761, EP0392225) is used as templatefor PCR with a left-to-right “topstrand” primer including a newlycreated ATG codon before the original GTG start codon, preceded by a GCCcodon, thus creating a NcoI restriction site at the ATG, and the first23 bases of the OtsA gene (primer TREB+: GTC AGC CAT GGT GAC AGA ACC GTTMC CGA MC, SEQ ID No:3) and a right-to-left “bottomstrand” primerhomologous to positions 181 to 205 downstream of the new ATG (primerTREB-; GTG CGT CAA GCT CCA CCA TTG AGC, SEQ ID No:4). This PCR reactionis undertaken with AmpliTaq DNA polymerase according to themanufacturer's recommendations (Perkin Elmer/Roche, Branchburg, N.J.)for five cycles at 94oC. (30 s), 4oC. (60 s), and 72oC. (30 s) followedby 25 cycles at 94° C. (30 s), 55° C. (60 s) and 72° C. (30 s) and thisgenerated a product of 212 bp containing a NcoI site at its left end anda EcoRV site at its right end. The fragment is gel purified usingstandard procedures, cleaved with NcoI and EcoRV and ligated into theNcoI and EcoRV sites of pUC21 to obtain pUCOTSB.

[0194] Plasmid pUCOTSB is then digested with SpeI and EcoRV, the 210 bpfragment containing the 5′ end of the OtsB gene is gel purified andligated with pCGN4467 that had previously been digested with XbaI andEcoRV to obtain pCGNOTSB containing the entire OtsB gene. PlasmidpCGNOTSB is digested with NcoI and SacI, the 0.8 kb long fragmentcontaining the OtsB gene is gel purified and ligated into the NcoI andSacI sites of pJG203 between a 903 bp long tobacco PR-1a promoter andthe nos gene termination signals, yielding pPR1OTSB containing the OtsBgene fused to the tobacco PR-1a promoter.

Example 3

[0195] Preparation of a Binary Vector Containing the OtsA Gene Fused tothe Tobacco PR-1a Promoter and the OtsB Gene Fused to the Tobacco PR-1aPromoter

[0196] Plasmid pPR1OTSA is digested with XhoI, the protruding ends arefilled-in with Klenow DNA polymerase (Promega, Madison, Wis.) and thenfurther digested with SpeI. The resulting 2.6 kb long fragment is gelpurified and ligated into the filled-in EcoRI site and the SpeI site ofpPR1OTSB to obtain pPR1OTSAB, containing the OtsA gene fused to thetobacco PR-1a promoter and the OtsB gene fused to the tobacco PR-1apromoter.

[0197] Plasmid pPR1OTSAB is digested with ApaI and XbaI, the 4.6 kb longfragment containing the OtsA gene fused to the tobacco PR-1a promoterand the OtsB gene fused to the tobacco PR-1a promoter is gel purifiedand ligated into the ApaI and XbaI sites of pBHYGM to obtain binaryvector pEGL502 (PBHYGM is a modified pGPTV-Hyg (Becker et al. (1992)Plant Mol. Biol. 20, 1195-1197) vector produced by insertion of apolylinker containingBfrI/ApaI//CIAI/SmaI/BfrI/XbaI/SaII/PstI/SphI/HindIII restriction sitesin the EcoRI and XbaI sites of pGPTV-Hyg).

Example

[0198] Preparation of a Binary Vector Containing the OtsA Gene Fused tothe Tobacco PR-1a Promoter

[0199] Plasmid pPR1OTSA is digested with ApaI and XbaI, the 2.6 kb longfragment containing the OtsA gene fused to the tobacco PR-1a promoter isgel purified and ligated into the ApaI and XbaI sites of pBHYGM toobtain a binary vector containing the OtsA gene fused to the tobaccoPR-1a promoter.

Example

[0200] Preparation of a Binary Vector Containing the OtsB Gene Fused tothe Tobacco PR-1a Promoter

[0201] Plasmid pPR1OTSB is digested with ApaI and XbaI, the 2.0 kb longfragment containing the OtsB gene fused to the tobacco PR-1a promoter isgel purified and ligated into the ApaI and XbaI sites of pBHYGM toobtain a binary vector containing the OtsB gene fused to the tobaccoPR-1a promoter.

Example 6

[0202] Transformation of Tobacco Leaf Discs by A. tumefaciens

[0203] The binary vector constructs are transformed into A. tumefaciensstrain GV3101 (Bechtold, N. et al. (1993), CR Acad. Sci. Paris, Sciencesde la vie, 316:1194-1199) by electroporation (Dower, W. J. (1987), Mol.Biol. Rep 1:5). Leaf discs of Nicotiana tabacum cv ‘Xanthi nc’ and oftransgenic line “NahG” overexpressing a salicylate hydroxylase gene(Gaffney et al. (1993) Science 261: 754-756) are cocultivated withAgrobacterium clones containing the above mentioned constructs (Horschet al. (1985), Science 227: 1229-1231) and transformants are selectedfor resistance to 50 mg/ml hygromycin B. Approximatively 50 independenthygromycin lines (T₀ lines) for each construct are selected and rootedon hormone-free medium.

Example 7

[0204] Selection of Transgenic Lines with Inducible trehaloseBiosynthetic Gene Expression

[0205] For each transgenic line a leaf punch of approximatively 2-3 cm²is incubated for 2 days in 3 ml of benzo(1,2,3)thiadiazole-7-carbothioicacid S-methyl ester (BTH, 5.6 mg/10 ml) under ca. 300 mmol/m⁻² s⁻¹irradiants. Leaf material is harvested, flash frozen and ground inliquid nitrogen. Total RNA is extracted (Verwoerd et al. (1989) NAR 17,2362) and Northern blot analysis is carried out as described (Ward etal. (1991) The Plant Cell 3, 1085-1094) using radiolabelled probesspecific for the OtsA and OtsB genes. Transgenic lines with highinducible expression of the trehalose biosynthetic genes in presence ofthe chemical inducer and low background expression in absence of thechemical inducer are selected. In particular, two transgenic lines areselected N5 and N6 and self-pollinated, and their progeny is used forfurther analysis.

Example 8

[0206] Transformation of Maize

[0207] The method used for maize transformation has been described byKoziel et al. (Biotechnology 11, 194-200, 1993) using particlebombardment into cells of immature embryos.Transformation of maize withat least one of the plasmids described herein is achieved bymicroprojectile bombardment of either immature zygotic embryos orserially-propagatable Type I embryogenic callus.

[0208] Type I embryogenic callus cultures (Green et al., Miami WinterSymposium 20,1983) of the proprietary genotype CG00526 and CG00714 areinitiated from immature embryos, 1.5-2.5 mm in length, from greenhousegrown material. Embryos are aseptically excised from surface-sterilizedears approximately 14 days after pollination. Embryos of CG00526 areplaced on D callus initiation media with 2% sucrose and 5 mg/Lchloramben (Duncan et al., Planta 165: 322-332,1985) while those ofCG00714 are placed onto KM callus initiation media with 3% sucrose and0.75 mg/L 2,4-d (Kao and Michayluk, Planta 126:105-110, 1975). Embryosand embryogenic cultures are subsequently cultured in the dark.Embryogenic responses are removed from the explants after ˜14 days.CG00526 responses are placed onto D callus maintenance media with 2%sucrose and 0.5 mg/L 2,4-d while those of CG00714 are placed onto KMcallus maintenance media with 2% sucrose and 5 mg/L Dicamba. After 3 to8 weeks of weekly selective subculture to fresh maintenance media, highquality compact embryogenic cultures are established. Actively growingembryogenic callus pieces are selected as target tissue for genedelivery. The callus pieces are plated onto target plates containingmaintenance medium with 12% sucrose approximately 4 hours prior to genedelivery. The callus pieces are arranged in circles, with radii of 8 and10 mm from the center of the target plate.

[0209] Plasmid DNA is precipitated onto gold microcarriers as describedin the DuPont Biolistics manual. Two to three μg of each plasmid is usedin each 6 shot microcarrier preparation. Genes are delivered to thetarget tissue cells using the PDS-100He Biolistics device. The settingson the Biolistics device are as follows: 8 mm between the rupture discand the macrocarrier, 10 mm between the macrocarrier and the stoppingscreen and 7 cm between the stopping screen and the target. Each targetplate is shot twice using 650 psi rupture discs. A 200×200 stainlesssteel mesh (McMaster-Carr, New Brunswick, N.J.) is placed between thestopping screen and the target tissue.

[0210] Seven days after gene delivery, target tissue pieces aretransferred from the high osmotic medium to high level selection media.All amino acids are removed from the selection media. After 5 to 8 weekson these high level selection media, any growing callus from CG00526 issubcultured to low to medium level media.

[0211] Tissue surviving selection from an original target tissue pieceis subcultured as a single colony and designated as an independenttransformation event.

[0212] At that point, colonies selected on selection media aretransferred to a modified MS medium (Murashige and Skoog, Physiol.Plant, 15:473-497, 1962) containing 3% sucrose (MS3S) with no selectionagent and placed in the light. For CG00526, 0.25 mg/L ancymidol and 0.5mg/L kinetin are added to this medium to induce embryo germination whilefor CGO0714, 2 mg/L benzyl adenine is added.

[0213] Regenerating colonies are transferred to MS3S media withoutancymidol and kinetin or benzyl adenine after 2 weeks. Regeneratingshoots with or without roots from all colonies are transferred toMagenta boxes containing MS3S medium and small plants with roots areeventually recovered and transferred to soil in the greenhouse.

[0214] Transformation events have also been created using Type I callusobtained from immature zygotic embryos using standard culturetechniques. For gene delivery, approximately 300 mg of the Type I callusis prepared by subculturing to fresh media 1 to 2 days prior to genedelivery, selecting target tissue pieces and placing them in a ringpattern 10 mm from the center of the target plate on medium againcontaining 12% sucrose. After approximately 4 hours, the tissue isbombarded using the PDS-1000/He Biolistic device from DuPont. Theplasmids are precipitated onto 1 um gold particles using the standardprotocol from DuPont. Genes are delivered using two shots per targetplate at 650 psi. Approximately 16 hours after gene delivery the callusis transferred to standard culture medium containing 2% sucrose with noselection agent. At 12 or 13 days after gene delivery, target tissuepieces are transferred to selection media containing 40 mg/lphosphinothricin as either Basta or bialaphos. The callus is subculturedon selection for 12 to 16 weeks, after which surviving and growingcallus is transferred to standard regeneration medium.

Example 9

[0215] Transformation of Wheat

[0216] Transformation of immature embryos and immature embryo-derivedcallus using particle bombardment has been described by Vasil et. al.(Biotechnology 11: 1553-1558,1993) and Weeks et. al. (Plant Physiology102: 1077-1084, 1993).

[0217] A preferred technique for wheat transformation involves particlebombardment of immature wheat embryos and includes either a high sucroseor a high maltose step prior to gene delivery. Prior to bombardment, anynumber of embryos (0.75-1 mm in length) are plated onto MS medium with3% sucrose (Murashige and Skoog, 1962) and 3 mg/l 2,4-D for induction ofsomatic embryos which is allowed to proceed in the dark. On the chosenday of bombardment, embryos are removed from the induction medium andplaced onto the osmoticum (i.e. induction medium with sucrose or maltoseadded at the desired concentration, typically 15%). The embryos areallowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryosper target plate is typical, although not critical. An appropriategene-carrying plasmid is precipitated onto micrometer size goldparticles using standard procedures. Each plate of embryos is shot withthe DuPont Biolistics helium device using a burst pressure of ˜1000 psiand using a standard 80 mesh screen. After bombardment, the embryos areplaced back into the dark to recover for about 24 h (still onosmoticum). After 24 hrs, the embryos are removed from the osmoticum andplaced back onto induction medium where they stay for about a monthbefore regeneration. Approximately one month later the embryo explantswith developing embryogenic callus are transferred to regenerationmedium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing theappropriate selection agent. After about one month, developed shoots aretransferred to larger sterile containers known as GA7s which containedhalf-strength MS, 2% sucrose, and the same concentration of selectionagent. The stable transformation of wheat is described in detail inpatent application EP 0 674 715.

Example 10

[0218] Transformation of Rice

[0219] Immature spikelets with milky endosperm of the Japonica ricevariety “Taipei 309” are dehulled and surface sterilized with 70% (v/v)ethanol for 1 min and 6% calcium hypochlorite for 20 min, followed bythree ishes with sterile distilled water.

[0220] The isolated immature embryos are cultured at 28° C. on 0.35%agarose-solidified MS-medium (Murashige and Skoog, 1962) containing 3%sucrose, 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.8. Afterone week, callus material produced from the scutella is divided andcultured by weekly transfers onto fresh medium. Four weeks after theinitiation, three to four calli are transferred into a 50-ml-culturevessel containing 20 ml of R2-medium (R2 salts and vitamins [Ohira etal. 1973], 1 mg/l 2,4-D, 500 mg/l 2-morpholino ethanesulfonic acid[MES], 3% sucrose, pH 5.8). The cultures are maintained in dim light at28° C. on a rotary shaker at 220 rpm, and the medium is replaced weeklyby an equal amount of fresh medium. Rapidly dividing, friable calli areselected and subcultured into a fresh container by transferring 2 ml offine callus suspension into 20 ml of R2-medium.

[0221] Two- to 3-month-old suspension cultures that have beensubcultured 3 to 4 days in advance serve as target cells for thebombardments. Four hours before particle bombardment, approx. 500 mg ofcells are spread as a single layer of 2 cm in diameter on 0.35%agarose-solidified piasmolysis medium (R2 salts and vitamins, 1 mg/l2,4-D, 3% sucrose, 0.5 M sucrose, pH 5.8) contained in a 5.5-cm petridish.

[0222] A particle inflow gun (Finer et al., 1992) is used to deliverDNA-coated gold particles (Aldrich Cat. # 32,658-5, spherical goldpowder 1.5-3.0 μm) into the embryogenic suspension cells. Particlecoating is essentially performed as described by Vain et al. (1993): 5μl aliquots of the plasmid solution are distributed into 0.5 ml-reactiontubes and placed on ice. Particles are suspended in 96% ethanol at 100mg/ml and vortexed for 2 min. Ethanol is replaced by an equal volume ofsterile ddH₂O and the suspension vortexed for 1 min. This washing stephas to be repeated once. The particles are finally resuspended insterile ddH₂O at 100 mg/ml. 25 μl of the particle suspension are addedto each of the DNA aliquots and the tubes vortexed for 1 min, followedby immediate addition of 25 μl of sterile, ice-cold CaCl₂ (2.5 M inddH₂O) and further vortexing for 1 min. 10 μl of sterile spermidine (0.1M in ddH₂O) are added, the suspension vortexed again and placed on icefor 5 min during which the particles sediment. 50 μl of theparticle-free supernatant are removed and the remaining suspension (15μl) used for 5 bombardments. Prior to each bombardment, the particlesneed to be resuspended by intense pipetting.

[0223] The cells are covered with a 500 μm mesh baffle and positioned at14 cm below the filter unit containing the particles. Particles arereleased by a single 8-bar-pressure pulse of 50 msec in partial vacuum(2×10⁴ Pa).

[0224] 24 h post bombardment with one of the transformation vectorsmentioned, the cells are transferred onto 0.3% agarose-solidified,selective callus increasing medium R2l (R2 salts, 1 mg/l 2,4-D, 1 mg/lthiamine-HCl, 500 mg/l MES, 6% sucrose, pH 5.8) containing a suitableselection agent such as, for example, 30 mg/l paromomycin, andmaintained at 280° C. in darkness for 3 weeks until theparomomycin-resistant (Pam^(R)) colonies become visible under the stereomicroscope. Pam^(R) colonies are transferred onto fresh R2l mediumcontaining 40 mg/l paromomycin and cultured in darkness (weeklysubculture). After 2 weeks, Pam^(R) colonies are transferred to 0.5%agarose-solidified R2l containing 40 mg/l paromomycin and cultured for 1week in darkness. For regeneration, colonies are then transferred onto0.8% agarose-solidified shoot induction medium (R2R: R2 salts, MSvitamins, 2% sucrose, 3% sorbitol, 1 mg/l zeatin, 0.5 mg/l IAA, 40 mg/lparomomycin) and cultured in light until shoots are formed. In parallel,callus material is maintained on R2l medium containing 40 mg/lparomomycin and cultured in darkness with weekly subcultures in order toobtain homoplasmic cell lines.

[0225] Expression of the Trehalose-6-phosphate Synthase andTrehalose-6-phosphate Phosphatase Genes in the Plant Plastid

[0226] B1. Inducible Expression

Example 11

[0227] Construction of Vector pAT236 for Homologous Recombination intothe Plastid Genome

[0228] The tmV and rps12/7 intergenic region of the tobacco plastidgenome is modified for insertion of chimeric genes by homologousrecombination. A 1.78 kb region (positions 139255 to 141036, Shinozakiet al. (1986) EMBO J. 5:2043-2049) is PCR amplified from the tobaccoplastid genome and a PstI site is inserted after position 140169,yielding 915 bp and 867 bp of flanking plastid DNA 5′ and 3′ of the PstIinsertion site. PCR amplification (Pfu Turbo DNA Polymerase, Stratagene,La Jolla, Calif.) is performed with a primer pair inserting a BsiEI sitebefore position 139255 (5′-TAA CGG CCG CGC CCA ATC ATT CCG GAT A-3′, SEQID No:5) and a PstI site after position 140169 (5′-TAA CTG CAG AAA GAAGGC CCG GCT CCA A-3′, SEQ ID No:6). PCR amplification is also performedwith a primer pair inserting a PstI site before position 140170 (5′-CGCCTG CAG TCG CAC TAT TAC GGA TAT G-3′, SEQ ID No:7) and a BsiWI siteafter position 141036 (5′-CGC CGT ACG AAA TCC TTC CCG ATA CCT C-3′, SEQID No:8). The PstI—BsiEI fragment is inserted into the PstI-SacII sitesof pbluescript SK+(Stratagene), yielding pAT216 and the PstI-BsiWIfragment is inserted into the PstI-Acc651 sites of pbluescript SK+,yielding pAT215. PAT218 contains the 1.78 kb of plastid DNA with a PstIsite for insertion of chimeric genes and selectable markers and isconstructed by ligation of the 2.0 kb PstI-ScaI fragment of pAT215 andthe 2.7 kb PstI-ScaI band of pAT216.

[0229] I. Amplification of the Tobacco 16S rRNA Promoter and rbs of therbcL Gene

[0230] The 16S rRNA promoter is PCR amplified from tobacco DNA (N.tabacum cv. Xanthi) and fused to a synthetic ribosome binding site (rbs)of the tobacco plastid rbcL gene. The “top strand” primer inserts anEcoRI site at the 5′ end of the 16S rRNA promoter before position 102568(5′-GCC AGA ATT CGC CGT CGT TCA ATG AGA ATG-3′, SEQ ID NO:9). The bottomstrand” primer amplifies up to position 102675 of the 16S rRNA promoter,removes two upstream ATG's by changing positions 102661 (A to C) and102670 (A to C), adds the rbs of the rbcL gene (positions 57569-57584)as a 5′ extension of the primer and inserts a BspHI site at the 3′ endof the rbs (5′-GCC TTC ATG ATC CCT CCC TAC AAC TAT CCA GGC GCT TCA GATTCG-3′, SEQ ID NO:10). The 142 bp amplification product is gel purifiedand cleavage with EcoRI and BspHI yields a 128bp fragment containing thetobacco 16S rRNA promoter fused to the rbs of the rbcL gene.

[0231] II. Amplification of the Tobacco Plastid rps16 Gene 3′Untranslated RNA Sequence (3′UTR)

[0232] The tobacco plastid rps16 3′ UTR is PCR amplified from tobaccoDNA (N. tabacum cv. Xanthi) using the following oligonucleotide pair: aSpeI site is added immediately after the stop codon of the plastid rps16gene encoding ribosomal protein S16 with the “top strand” primer (5′ CGCGAC TAG TTC AAC CGA AAT TCA AT-3′, SEQ ID NO: 11) and a PstI site isadded at the 3′ end of the rps16 3′ UTR with the “bottom strand” primer(5′-CGC TCT GCA GTT CAA TGG AAG CAA TG-3′, SEQ ID NO:12). Theamplification product is gel purified and digested with SpeI and PstI,yielding a 163 bp fragment containing the tobacco rps6 3′ UTR (positions4941 to 5093 of the tobacco plastid genome, Shinozaki et al., 1986)flanked 5′ by a SpeI site and 3′ with a PstI site.

[0233] III. Construction of a 16S rRNA Promoter:: aadA Gene:: rps163′UTR cassette for plastid transformation selection

[0234] The coding sequence of the aadA gene, a bacterial gene encodingthe enzyme aminoglycoside 3″ adenyltransferase that confers resistanceto spectinomycin and streptomycin, is isolated from pRL277 (Black et al.(1993) Molecular Microbiology 9:77-84 and Prentki et al. (1991) Gene103: 17-23). The 5′ major portion of the aadA coding sequence isisolated as a 724 bp BspHI-BssHII fragment from pRL277 (the startingcodon is at the BspHI site) and the 3′ remainder of the aadA gene ismodified by adding a SpeI site 20 bp after the stop codon by PCRamplification using pRL277 as template and the following oligonucleotidepair: the “top strand” primer (5′-ACC GTA AGG CTT GAT GAA-3′, SEQ IDNO:13) and the “bottom strand” primer which add a SpeI site (5′-CCC ACTAGT TTGAAC GAA TTG TTA GAC-3′, SEQ ID NO:14). The 658 bp amplificationproduct is gel purified, digested with BssHII, SpeI and the 89 bpfragment is ligated to the 5′ portion of the aadA gene carried on a 724bp BspHI-BssHII fragment, the 16S rRNA promoter and rbs of rbcL carriedon a 128 bp EcoRI-BspHI PCR amplified fragment and EcoRI-SpeI digestedpLITMUS28 vector (New England Biolabs), yielding pAT223. A three-wayligation is performed on an EcoRI-SpeI 0.94 kb fragment of pAT223containing the 16S rRNA promoter-rbs driven aadA gene, a 163 bp SpeI,PstI digested PCR fragment containing the rps16 3′ UTR and pucl9 (NewEngland Biolabs) cut with EcoRI, PstI to obtain pAT229 containing the16S rRNA promoter driving the aadA gene with the rps16 3′UTR.

[0235] IV. Amplification of the Bacteriophage T7 Gene 10 Promoter

[0236] The bacteriophage T7 gene 10 promoter is PCR amplified frompET-3d (Statagene) using the following oligonucleotide pair: the “topstrand” primer inserted an EcoRI site at the 5′ end of the T7 promoter(5′-CCC GAA TTC ATC CCG CGA AAT TAA TA-3′, SEQ ID NO:15) and the “bottomstrand” primer inserted a NcoI site at the 3′ end (5′-CGG CCA TGG GTATAT CTC CTT CTT AAA GTT AAA-3′, SEQ ID NO:16). The amplification productis gel purified and cleavage with EcoRI, NcoI produces a 96 bp fragmentcontaining the T7 promoter.

[0237] V. Amplification of the Bacteriophage T7 Gene 10 Terminator

[0238] The bacteriophage T7 gene 10 terminator is PCR amplified frompET-3d (Stratagene) using the following oligonucleotide pair: the “topstrand” primer inserts a HindIII site at the 5′ end of the terminator(5′-GCG AAG CTT GCT GAG CAA TAA CTA GCA TAA-3′, SEQ ID NO:17) and the“bottom strand” primer inserts a PstI site at the 3′ end of theterminator (5′-GCG CTG CAG TCC GGA TAT AGT TCC TCC T-3′, SEQ ID NO:18).The amplification product is gel purified and cleavage with HindIII-PstIproduces a 86 bp fragment containing the T7 terminator.

[0239] VI. Amplification of the Arabidopsis Thaliana Plastid psbA 3′Untranslated RNA Sequence

[0240] The A. thaliana plastid psbA 3′ UTR is PCR amplified from A.thaliana DNA (ecotype Landsburg) using the following oligonucleotidepair: the “top strand” primer adds a SpeI site to the 5′ end of the 3′UTR and eliminates a XbaI site in the native sequence by mutating a G toan A (underlined) (5′-GCG ACT AGT TAG TGT TAG TCT AAA TCT AGT T-3′, SEQID NO:19) and the “bottom strand” primer adds a HindIII site to the 3′end of the UTR (5′-CCG CAA GCT TCT AAT AAA AAA TAT ATA GTA-3′, SEQ IDNO:20). The amplified region extends from position 1350 to 1552 ofGenBank accession number X79898. The 218 bp PCR product is gel purified,digested with SpeI and HindIII and ligated with the HindIII-PstI cut PCRfragment carrying the T7 terminator into the SpeI-PstI sites ofpbluescript sk−(Stratagene), yielding pPH171. Sequence analysis of thepsbA 3′ UTR region of pPH171 compared to GenBank accession number X79898reveals deletion of an A at positions 1440 and 1452.

[0241] VII. Preparation of a Chimeric Gene Containing the GUS ReporterGene Fused to a Bacteriophage T7 gene 10 Promoter and Terminator and theArabidopsis Plastid psbA 3′UTR in a plastid transformation vector

[0242] A bacteriophage T7 gene 10 promoter:: GUS gene:: A. thaliana psbA3′ UTR :: T7 terminator cassette is constructed with a four-way ligationof the 96 bp EcoRI, NcoI PCR fragment containing the T7 promoter, a 1.86kb NcoI, XbaI fragment from pC8 containing the GUS gene, and the 295 bpXbaI, PstI fragment of pPH171 containing the A. thaliana psbA 3′ UTR andT7 terminator into the EcoRI, PstI sites of pGEM-3Z (Stratagene),yielding plasmid pAT221. The T7 promoter driven GUS gene cassette isligated to the aadA selectable marker cassette by cloning the 1.1 kbHindIII, EcoRI fragment of pAT229 containing the 16S rRNA promoter-rbs:: aadA :: rps16 3′ UTR cassette and the 2.26 kb EcoRI, PstI pAT221fragment carrying the T7 promoter :: GUS :: psbA 3′ UTR :: T7 terminatorcassette into the HindIII, PstI sites of pbluescript sk+ (Stratagene),producing plasmid pAT232. Plastid transformation vector pAT236 isconstructed by ligating the 3.36 kb PstI band from pAT232 containing theGUS and selectable marker cassettes into the PstI site of pAT218 andscreening for an insert orientation where the GUS gene is transcribed inthe same direction as the rps12/7 ORF.

Example 12

[0243] Construction of a Vector Using a Polyguanosine Tract as aSubstitute for a 3′UTR

[0244] A polyguanosine tract has been shown to substitute for theplastid atpB gene 3′ UTR in vivo (Drager et al. (1996) RNA 2:652-663). Apoly G tract containing 18 consecutive guanosine residues flanked bySpeI, HindIII sticky ends on the 5′ and 3′ ends respectively isassembled by annealing the following two kinased oligonucleotides:(5′-CTA GTG GGG GGG GGG GGG GGG GGA-3′, SEQ ID NO:21) and (5′-AGC TTCCCC CCC CCC CCC CCC CCA-3′, SEQ ID NO:22). The polyG₁₈ tract containingSpeI, HindIII sticky ends is ligated with the HindIII, PstI digested PCRfragment containing the T7 terminator into the SpeI, PstI sites ofpBluescript SK+ (Stratagene).

Example 13

[0245] Preparation of a Chimeric Gene Containing the E. coliTrehalose-6-phosphate Synthase Gene (OtsA) Fused to the Phage T7 Gene 10Promoter in a Plastid Transformation Vector

[0246] Genomic DNA from E. coli strain DH5-alpha is used as template forPCR amplification of the 5′ portion of the OtsA gene with a top strandprimer incorporating the ATG start codon followed by a newly added GCAcodon, thus creating an NcoI restriction site (primer pOTSAN+: 5′-TGACCA TGG CAA GTC GTT TAG TCG TAG T-3′, SEQ ID NO:23), and a bottom strandprimer downstream of the unique SfuI restriction site in OtsA (pOTSAN-:5′-AGCAAC GCT TCA TAG-3′, SEQ ID NO:24). PCR reactions are undertaken in50 ul volumes using PFU DNA polymerase (Promega) as recommended by themanufacturer in a DNA Thermocycler 480 (Perkin Elmer/Roche, Branchburg,N.J.) for five cycles at 94° C. (30 s), 40° C. (60 s), and 72° C. (30 s)followed by 25 cycles at 94° C. (30 s), 55° C. (60 s) and 72° C. (30 s).The 850 bp PCR product is gel purified using standard procedures andcleaved with NcoI (all restriction enzymes obtained from New EnglandBiolabs except where otherwise noted) and SfuI (Boehringer Mannheim,Corp., Indianapolis) to release a 661 bp DNA fragment. The 3′ portion ofOtsA is obtained in a similar manner as described above using a topstrand primer (pOTSAX+: 5′-GCG TTC CTG GAT TGT C-3′, SEQ ID NO:25)located upstream of the SfuI site in OtsA, and a bottom strand primer(pOTSAX-: 5′-GGG TCT AGA GAT TCA CGC GAG CTT TGG AAA GGT AGC A-3′, SEQID NO:26) that introduces an XbaI restriction site downstream of thestop codon and destroys the HindIII restriction site present at the 3′end of OtsA by changing the CTT Leu codon to CTC. The 861 bpamplification product is gel purified, digested with SfuI and XbaI, andthe resulting 772 bp DNA fragment ligated with the 5′ OtsA NcoI/SfuIfragment in pLitmus28 (Promega) digested with NcoI and XbaI to createpOTSA.

[0247] Plasmid DNA from pAT236 (Example 11), containing a phage T7 gene10 promoter cassette from pET3a (Novagen) in a plastid transformationvector, is digested with NcoI and SphI (to create a 1646 bp fragment)and SphI and XbaI (to create a 4514 bp fragment). These vector fragmentsare ligated in a three-way reaction with the 1433 bp NcoII/XbaI fragmentof pOTSA that contains the complete OtsA gene to create plastidtransformation vector pT7-OTSA.

Example 14

[0248] Preparation of a Chimeric Gene Containing the E. coliTrehalose-6-phosphate Phosphatase Gene (OtsB) Fused to the Phage T7 Gene10 Promoter in a Plastid Transformation Vector

[0249] The 5′ portion of the OtsB gene is amplified from E. coli genomicDNA as described above using top strand primer pOTSBN+: 5′-GTC GCC ATGGTG ACA GAA CCG TTA ACC-3′, SEQ ID NO:27, that converts the GTG startcodon of OtsB to ATG and adds a GTG Val codon at the second position,and bottom strand primer pOTSBN-: 5′-GTT CGC CCG ATA AAG GGA G-3′, SEQID NO:28, located downstream of the unique BgIII site of OtsB. The 584bp product is gel-purified and digested with NcoI and BgIII and theresulting 459 bp fragment isolated. The 3′ portion of OtsB is similarlyamplified using top strand primer pOTSBX+: 5′-TAG CGCAAC GTA TTA CTC-3′,SEQ ID NO:29, located upstream of the OtsB BgIII site, and bottom strandprimer pOTSBX-: 5′-GCC TCT AGA CTC ATC ATT AGA TAC TAC GAC TAA AC-3′,SEQ ID NO:30, that incorporates an XbaI restriction site downstream ofthe OtsB stop codon. The gel-purified 381 bp product is digested withBgIII and XbaI, and the resulting 354 bp BgIII/XbaI restriction fragmentligated with the 5′ OtsB NcoI/BgIII restriction fragment into vectorpLitmus28 digested with NcoI and XbaI to create pOTSB.

[0250] Plasmid pOTSB is then digested with NcoI and XbaI and theresulting 820 bp fragment containing the complete OtsB gene is ligatedin a three-way reaction with the NcoI/SphI and SphI/XbaI fragments ofplasmid pAT236 as described above to create plastid transformationvector pT7_OTSB.

Example 15

[0251] Preparation of a Plastid Transformation Vector Containing anOperon-like Chimeric Gene Construct Containing the OtsA and the OtsBGenes Fused to a Bacteriophage T7 Promoter and Terminator

[0252] Plasmid pOTSB is digested with NcoI and SpeI and the 3534 bpvector backbone/OtsB fragment isolated and dephosphorylated. Thisfragment is ligated to a synthetic oligonucleotide linker containing aportion of the phage T7 gene 10 5′ UTR and a chimeric consensus plastidribosome binding site prepared by annealing and then phosphorylatingwith T4 kinase the top strand oligonucleotide 5′-CTA GTG GGA GAC CACAACGGT TTC CCT CTA GAA ATA ATT TTG TTT AAG TTT AAG AAG GGG AGA GAA T-3′,SEQ ID NO:31 (SpeI restriction site overhang underlined) and the bottomstrand oligonucleotide 5′-CAT GAT TCT CTC CCC TTC TTA AAC TTA AAC AAAATT ATT TCT AGA GGG AAA CCG TTG TGG TCT CCC A-3′, SEQ ID NO:32 (BspHIrestriction site overhang underlined). The resulting plasmid pOTSBL isdigested with SpeI and ligated to a 1516 bp SpeI/XbaI fragment of pOTSAselected for the orientation SpeI—OtsA::linker::OtsB to create pOTSABL.Plasmid pOTSABL is then digested with NcoI and XbaI (partial) and theresulting 2313 bp fragment containing the complete OtsA::T75′/RBS::OtsBcassette is ligated in a three-way reaction with the NcoI/SphI andSphII/XbaI fragments of plasmid pAT236 as described above to createplastid transformation vector pT7_OTSAB.

[0253] A similar plastid transformation vector comprising omitting theportion of the phage T7 gene 10 5′ UTR is also created using standardmethods in molecular biology.

[0254] B2. Constitutive Expression

Example 16

[0255] Amplification of the Tobacco Plastid cIpP Gene Promoter andComplete 5′ Untranslated RNA (5′UTR).

[0256] Total DNA from N. tabacum c.v. “Xanthi NC” is used as thetemplate for PCR with a left-to-right “top strand” primer comprising anintroduced EcoRI restriction site at position −197 relative to the ATGstart codon of the constitutively expressed plastid cIpP gene (primerPclp_P1a: 5′-GCG GAA TTC ATA CTT ATT TAT CAT TAG AAA G-3′ (SEQ IDNO:33); EcoRI restriction site underlined) and a right-to-left “bottomstrand” primer homologous to the region from −21 to −1 relative to theATG start codon of the cIpP promoter that incorporates an introducedNcoI restriction site at the start of translation (primer Pclp_P2b:5′-GCG CCA TGG TAA ATG AAA GAA AGA ACT AAA-3′ (SEQ ID NO:34); NcoIrestriction site underlined). This PCR reaction is undertaken with Pfuthermostable DNA polymerase (Stratagene, La Jolla Calif.) in a PerkinElmer Thermal Cycler 480 according to the manufacturer's recommendations(Perkin Elmer/Roche, Branchburg, N.J.) as follows: 7 min 95° C.,followed by 4 cycles of 1 min 95° C./2 min 43° C./1 min 72° C., then 25cycles of 1 min 95° C./2 min 55° C./1 min 72° C. The 213 bpamplification product comprising the promoter and 5′ untranslated regionof the cIpP gene containing an EcoRI site at its left end and an NcoIsite at its right end and corresponding to nucleotides 74700 to 74505 ofthe N. tabacum plastid DNA sequence (Shinozaki et al., EMBO J. 5:2043-2049 (1986)) is gel purified using standard procedures and digestedwith EcoRI and NcoI (all restriction enzymes are purchased from NewEngland Biolabs, Beverly, Mass.).

Example 17

[0257] Amplification of the Tobacco Plastid rps16 Gene 3′ UntranslatedRNA Sequence

[0258] Total DNA from N. tabacum c.v. “Xanthi NC” is used as thetemplate for PCR as described above with a left-to-right “top strand”primer comprising an introduced XbaI restriction site immediatelyfollowing the TAA stop codon of the plastid rps16 gene encodingribosomal protein S16 (primer rps16P_(—)1a (5′-GCG TCT AGA TCA ACC GAAATT CAA TTA AGG-3′ (SEQ ID NO:35); XbaI restriction site underlined) anda right-to-left “bottom strand” primer homologous to the region from+134 to +151 relative to the TAA stop codon of rps16 that incorporatesan introduced HindIII restriction site at the 3′ end of the rps16 3′ UTR(primer rps16P_(—)1b (5′-CGC AAG CTT CAA TGG AAG CAA TGA TAA-3′ (SEQ IDNO:36); HindIII restriction site underlined). The 169 bp amplificationproduct comprising the 3′ untranslated region of the rps16 genecontaining an XbaI site at its left end and a HindIII site at its rightend and containing the region corresponding to nucleotides 4943 to 5093of the N. tabacum plastid DNA sequence (Shinozaki et al., 1986) is gelpurified and digested with XbaI and HindIII.

Example 18

[0259] Preparation of a Plastid Transformation Vector Containing a GUSReporter Gene Fragment Ligated to the cipP Gene Promoter and 5′ and 3′UTR's.

[0260] An 1864 bp b-galacturonidase (GUS) reporter gene fragment derivedfrom plasmid pRAJ275 (Clontech) containing an NcoI restriction site atthe ATG start codon and an XbaI site following the native 3′ UTR isproduced by digestion with NcoI and XbaI. This fragment is ligated in afour-way reaction to the 201 bp EcoRI/NcoI cipP promoter fragment, the157 bp XbaI/HindIII rps16 3′UTR fragment, and a 3148 bp EcoRI/HindIIIfragment from cloning vector pGEM3Zf(-) (Promega, Madison Wis.) toconstruct plasmid pPH138. Plastid transformation vector pPH140 isconstructed by digesting plasmid pPRV111a (Zoubenko et al. (1994)Nucleic Acids Res 22:3819-24) with EcoRI and HindIII and ligating theresulting 7287 bp fragment to a 2222 bp EcoRI/HindIII fragment ofpPH138.

Example 19

[0261] Preparation of a Plastid Transformation Vector Containing theOtsA Gene Ligated to the clpP Gene Promoter and 5′ and 3′ UTR's.

[0262] A 1433 bp NcoI/XbaI fragment of pOTSA that contains the completeOtsA gene is ligated in a four-way reaction to the 201 bp EcoRI/NcoIcIpP promoter fragment, the 157 bp XbaI/HindIII rps16 3′UTR fragment,and a 3148 bp EcoRI/HindIII fragment from cloning vector pGEM3Zf(−)(Promega, Madison Wis.) to construct plasmid pcIpOtsA. A plastidtransformation vector is constructed by digesting plasmid pPRV111a withEcoRI and HindIII and ligating the resulting 7287 bp fragment to a 1791bp EcoRI/HindIII fragment of pcIpOtsA.

Example 20

[0263] Preparation of a Plastid Transformation Vector Containing theOtsB Gene Ligated to the clpP Gene Promoter and 5′ and 3′ UTR's.

[0264] Plasmid pOTSB is digested with NcoI and XbaI and the resulting820 bp fragment containing the complete OtsB gene is ligated in afour-way reaction to the 201 bp EcoRI/NcoI cIpP promoter fragment, the157 bp XbaI/HindIII rps16 3′ UTR fragment, and a 3148 bp EcoRI/HindIIIfragment from cloning vector pGEM3Zf(−) (Promega, Madison Wis.) toconstruct plasmid pcIpOtsB. A plastid transformation vector isconstructed by digesting plasmid pPRV111a with EcoRI and HindIII andligating the resulting 7287 bp fragment to a 1178 bp EcoRI/HindIIIfragment of pcIpOtsB.

Example 21

[0265] Preparation of a Plastid Transformation Vector Containing anOperon-like Chimeric Gene Construct Containing the OtsA Gene and OtsBGene Ligated to the clpP Gene Promoter and 5′ and 3′ UTR's.

[0266] Plasmid pOTSABL is digested with NcoI and XbaI (partial) and theresulting 2313 bp fragment containing the completeOtsA::T75′/RBS::OtsBcassette is ligated in a four-way reaction to the201 bp EcoRI/NcoI cIpP promoter fragment, the 157 bp XbaI/HindIII rps163′UTR fragment, and a 3148 bp EcoRI/HindIII fragment from cloning vectorpGEM3Zf(−) (Promega, Madison Wis.) to construct plasmid pcIpOtsAB.Plastid transformation vector pPH140 is constructed by digesting plasmidpPRV111a (Zoubenko et al. 1994) with EcoRI and HindIII and ligating theresulting 7287 bp fragment to a 2671 bp EcoRI/HindIII fragment ofpcIpOtsAB.

Example 22

[0267] Biolistic Transformation of the Tobacco Plastid Genome

[0268] Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated sevenper plate in a 1″ circular array on T agar medium and bombarded 12-14days after sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pC8E5 and pC+E5 essentially asdescribed (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombardedseedlings are incubated on T medium for two days after which leaves areexcised and placed abaxial side up in bright light (350-500 μmolphotons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentare subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones is assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with ³²P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompC8 containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) andtransferred to the greenhouse.

Example 23

[0269] Preparation of Transgenic Tobacco Expressing a ChemicallyInducible, Plastid-targeted T7 RNA Polymerase

[0270] A synthetic oligonucleotide linker comprising an NcoI restrictionsite and ATG start codon followed by the first seven plastid transitpeptide codons from the rbcS gene (encoding the small subunit ofribulose bisphosphate carboxylase) and endogenous PstI restriction site(top strand: 5′-CAT GGC TTC CTC AGT TCT TTC CTC TGC A-3′, SEQ ID NO:37;bottom strand: 5′-GAG GAA AGA ACT GAG GAA GC-3′, SEQ ID NO:38), a 2.8 kbPstI/SacI DNA fragment of pCGN4205 (McBride, K. E. et al. (1994) PNAS91, 7301-7305) containing the bacteriophage T7 RNA polymerase gene (T7Pol) fused in frame to the 3′ portion of the rbcS gene transit peptidecoding sequence, a 0.9 kb NcoI/KpnI DNA fragment of pCIB296 containingthe tobacco PR-1a promoter with an introduced NcoI restriction site atthe start codon (Uknes et al. (1993) Plant Cell 5, 159-169) and 4.9 kbSfiI/KpnI and 6.6 kb SacI/SfiI fragments of binary Agrobacteriumtransformation vector pSGCGC1 (a derivative of pGPTV-Hyg containing thepolylinker from pGEM4 (Promega, Madison Wis.) cloned into theSacI/HindIII sites) are ligated to construct pPH110 .

[0271] Hygromycin resistant NT-pPH110 tobacco plants are regenerated asdescribed from shoots obtained following cocultivation of leaf disks ofN. tabacum ‘Xanthi’ and “NahG” with GV3101 Agrobacterium carrying thepPH110 binary vector. For each transgenic line duplicate leaf punches ofapproximately 2-3 cm² are incubated for 2 days in 3 ml of BTH (5.6 mg/10ml) or sterile distilled water under ca. 300 μmol/m²/s irradiance. Leafmaterial is harvested, flash frozen and ground in liquid nitrogen. TotalRNA is extracted (Verwoerd et al. (1989) NAR 17, 2362) and Northern blotanalysis is carried out as described (Ward et al. (1991) The Plant Cell3, 1085-1094) using a radiolabelled T7 RNA polymerase gene probe. Plantsof nineteen NT-110X (Xanthi genetic background) and seven NT-11ON (NahGgenetic background) T1 lines showing a range of T7 Pol expression aretransferred to the greenhouse and self pollinated. Progeny segregating3:1 for the linked hygromycin resistance marker are selfed andhomozygous T2 lines selected.

Example 24

[0272] Plastid Transformation of Maize Type I embryogenic calluscultures (Green et al. (1983) in A. Fazelahmad, K. Downey, J. Schultz,R. W. Voellmy, eds. Advances in Gene Technology: Molecular Genetics ofPlants and Animals. Miami Winter Symposium Series, Vol. 20. AcademicPress, N.Y.) of the proprietary genotypes CG00526 and CG00714 areinitiated from immature embryos, 1.5-2.5 mm in length, from greenhousegrown material. Embryos are aseptically excised from surface-sterilizedears approximately 14 days after pollination. Embryos of CG00526 areplaced on D callus initiation media with 2% sucrose and 5 mg/Lchloramben (Duncan et al. (1985) Planta 165: 322-332) while those ofCG00714 are placed onto KM callus initiation media with 3% sucrose and0.75 mg/L 2,4-d (Kao and Michayluk (1975) Planta 126, 105-110). Embryosand embryogenic cultures are subsequently cultured in the dark.Embryogenic responses are removed from the explants after ˜14 days.CG00526 responses are placed onto D callus maintenance media with 2%sucrose and 0.5 mg/L 2,4-d while those of CG00714 are placed onto KMcallus maintenance media with 2% sucrose and 5 mg/L Dicamba. After 3 to8 weeks of weekly selective subculture to fresh maintenance media, highquality compact embryogenic cultures are established. Actively growingembryogenic callus pieces are selected as target tissue for genedelivery. The callus pieces are plated onto target plates containingmaintenance medium with 12% sucrose approximately 4 hours prior to genedelivery. The callus pieces are arranged in circles, with radii of 8 and10 mm from the center of the target plate. Plasmid DNA is precipitatedonto gold microcarriers as described in the DuPont Biolistics manual.Two to three μg of each plasmid is used in each 6 shot microcarrierpreparation. Genes are delivered to the target tissue cells using thePDS-1000He Biolistics device. The settings on the Biolistics device areas follows: 8 mm between the rupture disc and the macrocarrier, 10 mmbetween the macrocarrier and the stopping screen and 7 cm between thestopping screen and the target. Each target plate is shot twice using650 psi rupture discs. A 200×200 stainless steel mesh (McMaster-Carr,New Brunswick, N.J.) is placed between the stopping screen and thetarget tissue.

[0273] Five days later, the bombed callus pieces are transferred tomaintenance medium with 2% sucrose and 0.5 mg/L 2,4-d, but without aminoacids, and containing 750 or 1000 nM Formula XVII. The callus pieces areplaced for 1 hour on the light shelf 4-5 hours after transfer or on thenext day, and stored in the dark at 27° C.for 5-6 weeks. Following the5-6 week primary selection stage, yellow to white tissue is transferredto fresh plates containing the same medium supplemented with 500 or 750nM Formula XVII. 4-5 hours after transfer or on the next day, thetissues are placed for 1 hour on the light shelf and stored in the darkat 27° C. for 3-4 weeks. Following the 3-4 week secondary selectionstage, the tissues are transferred to plates containing the same mediumsupplemented with 500 nM Formula XVII. Healthy growing tissue is placedfor 1 hour on the light shelf and stored in the dark at 27° C. It issubcultured every two weeks until the colonies are large enough forregeneration.

[0274] At that point, colonies are transferred to a modified MS medium(Murashige and Skoog (1962) Physiol. Plant 15: 473-497) containing 3%sucrose (MS3S) with no selection agent and placed in the light. ForCG00526, 0.25 mg/L ancymidol and 0.5 mg/L kinetin are added to thismedium to induce embryo germination, while for CG00714, 2 mg/L benzyladenine is added. Regenerating colonies are transferred to MS3S mediawithout ancymidol and kinetin, or benzyl adenine, for CG00526 orCG00714, respectively, after 2 weeks. Regenerating shoots with orwithout roots are transferred to boxes containing MS3S medium and smallplants with roots are eventually recovered and transferred to soil inthe greenhouse.

[0275] C. Chemical Induction of the Trehalose Biosynthetic Genes andMeasurement of Trehalose Content in Plants

Example 25

[0276] Chemical Induction of the Trehalose Biosynthetic Genes

[0277] Seeds are germinated and plants are grown for 3-6 weeks in thegreenhouse. They are then sprayed with 1.2 mM BTH (or as furtherillustrated in Friedrich et al. (1996) Plant J. 10, 61-70) or withwettable powder. Samples of plant material are harvested at differenttime points and flash frozen. Northern Blot analysis is carried out tomonitor induction of expression of the trehalose biosynthetic genes upontreatment with BTH.

Example 26

[0278] Extraction of Soluble Sugars and Polyols from Lyophilized TobaccoTissue

[0279] 10-20 mg lyophilized tissue is extracted 3 times with 400 ml 80%methanol at 65° C. for 10 minutes after addition of 40 mg manoheptulose(internal standard). The combined supernatant (after centrifugation at13000 rpm for 5 minutes in an Eppendorff table top centrifuge) is driedunder vacuum in a Speedvac at 25° C. The dried extract is thenresuspended in 700 ml milipor water and desalted by adding 50 ml of amixed bed ion exchange resin (Serdolit micro blue and red 2:1 [v/v]).The ion exchange resin is sedimented by centrifugation at 13000 rpm andwashed with 300 ml Millipore water. The combined supernatant is againdried under vacuum in a Speedvac at 25° C. The residue containing mainlysugars and polyols is now ready for analysis by HPLC or forderivatisation for the subsequent analysis by capillary GC.

Example 27

[0280] HPLC Analysis

[0281] The dried residue is resuspended in 200 ml water and centrifugedfor 15 min at 15000 rpm. An aliquot of 10 ml is separated isocraticallywith a 100 mM NaOH solution on a Dionex P100 ion exchange column usingan HPLC system from Dionex equipped with a pulsed amperometric detector.

Example 28

[0282] Capillary GC

[0283] The dried residue is resuspended in 200 ml 50% methanol andcentrifuged for 15 min at 15000 rpm. 80 ml of the supernatant aretransferred into 200 ml GC injection vials. The sugars and polyols aredried under vacuum in a Speedvac. The residue is then rendered anhydrousby repeated evaporation of added methanol on a heating block at 80° C.The anhydrous residue is now sealed with septa containing screw caps.The samples are then dissolved in anhydrous pyridine containing 625 mghydroxylamine and 50 mg phenyl-b-glucopyranoside. This mixture isincubated at 80° C. for 30 minutes. After addition of 50 mlN-methyl-N-trimethylsilyl-heptafluoro-butyramide containing 1%trimethylchlorosilane (v/v) the derivatisation reaction is carried outfor 30 minutes at 80° C. The TMS-(trimethylsilyi)-derivatives of sugarsand polyols are now ready for analysis by GC. The separation of 1 to 3ml of this reaction mixture is performed with a Carlo Erba GC equippedwith a FID detector using the conditions listed below: Capillary: SWScientific, 30 m,ID 0.323 mm, liquid Phase DB-17. Temperature program:70° C., 2 min, 25° C./min to 170° C., 700° C./min to 340° C., 340° C., 5min

Example 29

[0284] Determination of the Trehalose Content in Transgenic Plants byHPLC

[0285] The progeny of two independent transgenic lines (N5/3 and N5/4for transgenic line N5, N6/1, N6/2, N6/7 and N6/8 for transgenic lineN6) are grown and treated with BTH as described in example 25. Samplesare harvested and extracted as described in example 26. The trehalosecontent is determined by HPLC (example 27).

[0286] Table 1 shows the trehalose contents of samples after BTHtreatment or after treatment with wettable powder (WP) as a control.Measurements of the trehalose content the wild-type Xanthi are alsoshown. The values are expressed in mg/g dry weight (DW) of the measuredsample. While no trehalose is detected in the wild-type Xanthi and intransgenic plants at day 0 or after treatment with BTH, trehalose isdetected after BTH treatment. sucrose trehalose glucose fructose (mg/g(mg/g DW) (mg/g DW) (mg/g DW) DW) Xanthi 0 day BTH 0 18.1 3.7 17.6Xanthi 3 days BTH 0 6.7 1.5 9.1 Xanthi 7 days BTH 0 11.9 2.2 16.4 N5/3 0day BTH 0 3 0.6 8.2 N5/3 3 days BTH traces 2.4 0.2 7 N5/3 7 days BTH 0.58.4 1.6 19.5 N5/3 7 days WP 0 6.9 1.5 12.9 N5/4 0 day BTH 0 7.3 1.8 16.7N5/4 3 days BTH traces 1.3 0.4 7.7 N5/4 7 days BTH 0.6 9.5 2.2 19.7 N5/422 days BTH 2.3 24 4.7 17.6 N5/4 7 days WP 0 24.9 4 10.1 N6/1 0 day BTH0 9.7 1.9 18.1 N6/1 3 days BTH 0 1.7 0.5 10.3 N6/1 7 days BTH traces10.7 2.4 23.6 N6/1 22 days BTH 0.7 31.7 6.2 13.2 N6/2 0 day BTH 0 3.60.6 11.1 N6/2 3 days BTH 0 4 1.1 8.6 N6/2 7 days BTH traces 6.1 1.3 20.1N6/7 0 day BTH 0 2.1 0.4 13.6 N6/7 3 days BTH 0.1 1.5 0.4 9.1 N6/7 7days BTH 1.2 12.7 2.9 25.8 N6/7 22 days BTH 2.6 37 6.5 17.1 N6/8 0 dayBTH 0 4 0.8 10.4 N6/8 3 days BTH 0 2.4 0.4 7.9 N6/8 7 days BTH 0.2 14.93.3 16.1

Example 30

[0287] Determination of Trehalose Content in Transgenic Plants byHPLC/GC

[0288] The progeny of two independent transgenic lines (N5/3 and N5/4for transgenic line N5, N6/1, N6/2, N6/7 and N6/8 for transgenic lineN6) are grown and treated with BTH as described in example 25. Samplesare harvested and extracted as described in example 26. The trehalosecontent is determined by HPLC/GC (example 28).

[0289] Table 2 shows the trehalose contents of samples after BTHtreatment or after treatment with wettable powder (WP) as a control.Measurements of the trehalose content the wild-type Xanthi are alsoshown. The values are expressed in mg/g dry weight (DW) of the measuredsample. Induction of trehalose accumulation in the transgenic plantafter BTH treatment is observed. mg Manoheptulose extracted TrehaloseGlucose Fructose Sucrose Mannitol Inositol (Int. Std. 40 pg) Xanthi 0day BTH 5.6 area 0 130626 137761 122625 0 50850 98724 mg/g DW 0 8.119.24 13.99 0 316 Xanthi 3 days BTH 5.5 area 0 23971 26108 50252 0 6745848803 mg/g DW 0 3.06 3.61 11.61 0 9.18 Xanthi 7 days BTH 8.7 area 0103175 86735 252532 0 156339 78786 mg/g DW 0 5.16 4.69 23.24 0 8.33 N5/30 day BTH 4.6 area 0 25664 26357 42743 2139 23091 76831 mg/g DW 0 2.492.82 7.63 0.23 2.39 N5/3 3 days BTH 8.3 area 2522 32522 25992 91395 255685370 78343 mg/g DW 0.25 1.72 1.48 8.87 0.15 4.8 N5/3 7 days BTH 10.6area 7341 115732 109582 461342 10312 156336 89033 mg/g DW 0.49 4.21 4.330.84 0.42 6.05 N5/3 7 days WP 6.2 area 1053 46576 50861 106758 432991251 81008 mg/g DW 0.13 3.18 3.75 13.41 0.33 6.64 N5/4 0 day BTH 6 area124 63822 75278 136497 6640 46626 98401 mg/g DW 0.01 3.71 4.73 14.590.43 2.88 N5/4 3 days BTH 6.7 area 1737 32083 34104 120958 6740 128468103019 mg/g DW 0.16 1.59 1.83 11.06 0.37 6.8 N5/4 7 days BTH 6.2 area3518 71221 83701 255283 3762 115824 97553 mg/g DW 0.37 4.04 5.13 26.630.24 7 N5/4 22 days BTH 10 area 17725 250258 265768 360298 14192 206874107392 mg/g DW 1.04 7.99 9.17 21.17 0.5 7.04 N5/4 7 days WP 7.9 area1266 230792 189040 149745 8927 134969 102634 mg/g DW 0.1 9.76 8.64 11.650.42 6.08 N6/1 0 day BTH 5.9 area 68 104570 109361 155436 6623 40609108035 mg/g DW 0.01 5.63 6.36 15.39 0.4 2.33 N6/1 3 days BTH 9.6 area171 28576 32618 189668 7263 203269 99197 mg/g DW 0.01 1.03 1.27 12.570.29 7.8 N6/1 7 days BTH 8.4 area 718 93789 109060 390133 7781 177790113543 mg/g DW 0.05 3.37 4.24 25-81 0.31 6.81 N6/1 22 days BTH 10.8 area3012 633512 599460 426678 15985 386790 124721 mg/g DW 0.14 16.13 16.519.99 0.45 10.49 N6/2 0 day BTH 5.7 area 0 31891 33610 73092 6854 2132469201 mg/g DW 0 2.77 3.16 11.69 0.66 1.97 N6/2 3 days BTH 6.8 area 021575 35235 82293 9340 90009 78756 mg/g DW 0 1.38 2.44 9.69 0.66 6.14N6/2 7 days BTH 8.5 area 447 47653 53238 290368 9295 105943 74862 mg/gDW 0.04 2.57 3.1 28.79 0.56 6.08 N6/7 0 day BTH 4.8 area 158 51929 46536102388 8893 63254 108621 mg/g DW 0.02 3.42 3.31 12.39 0.65 4.43 N6/7 3days BTH 4.9 area 2261 10420 13317 139589 3870 120264 117476 mg/g DW0.25 0.62 0.86 15.3 0.26 7.63 N6/7 7 days BTH 8 area 7813 107748 116835464737 0 179686 102089 mg/g DW 0.61 4.53 5.3 35.9 0 8.04 N6/7 22 daysBTH 14.1 area 31445 609847 642550 761211 24765 601151 122289 mg/g DW1.15 12.13 13.81 27.85 0.55 12.74 N6/8 0 day BTH 6.8 area 40 49010 51848111978 6328 26476 76039 mg/g DW 0 3.25 3.72 13.66 0.47 1.87 N6/8 3 daysBTH 6.1 area 447 12368 16802 74009 7182 117838 83366 mg/g DW 0.06 0.831.22 9.18 0.54 8.46 N6/8 7 days BTH 7.9 area 3051 147316 164133 2977605728 125094 89794 mg/g DW 0.27 7.12 8.58 26.48 0.31 6.44

Example 31

[0290] Determination of the Inducible Drought-resistance of TransgenicPlants

[0291] Seven days after treatment with BTH or wettable powder (seeexample 25), the plants are taken off the irrigation system and notwatered any longer. They are further grown and their phenotype ismonitored. Fourteen days later BTH-treated plants had grown further andlooked like irrigated control plants, whereas plants treated withwettable powder are completely dessicated. BTH-treated plants are grownfurther and are allowed to set seeds. Drought resistance thereforecorrelates with the expression of the trehalose biosynthetic genes andthe accumulation of trehalose.

[0292] The above disclosed embodiments are illustrative. This disclosureof the invention will place one skilled in the art in possession of manyvariations of the invention. All such obvious and foreseeable variationsare intended to be encompassed by the appended claims.

1 38 1 37 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 1 gtcagccatg gcaagtcgtt tagtcgtagt atctaac 37 2 24 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide 2gcaaatggca acaggtgata atcg 24 3 33 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide 3 gtcagccatg gtgacagaaccgttaaccga aac 33 4 24 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide 4 gtgcgtcaag ctccaccatt gagc 24 5 28 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide 5taacggccgc gcccaatcat tccggata 28 6 28 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 6 taactgcagaaagaaggccc ggctccaa 28 7 28 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 7 cgcctgcagt cgcactatta cggatatg 288 28 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 8 cgccgtacga aatccttccc gatacctc 28 9 30 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 9 gccagaattcgccgtcgttc aatgagaatg 30 10 45 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 10 gccttcatga tccctcccta caactatccaggcgcttcag attcg 45 11 26 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 11 cgcgactagt tcaaccgaaa ttcaat 2612 26 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 12 cgctctgcag ttcaatggaa gcaatg 26 13 18 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 13accgtaaggc ttgatgaa 18 14 27 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 14 cccactagtt tgaacgaatt gttagac 2715 26 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 15 cccgaattca tcccgcgaaa ttaata 26 16 33 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 16cggccatggg tatatctcct tcttaaagtt aaa 33 17 30 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 17 gcgaagcttgctgagcaata actagcataa 30 18 28 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 18 gcgctgcagt ccggatatag ttcctcct 2819 31 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 19 gcgactagtt agtgttagtc taaatctagt t 31 20 30 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide20 ccgcaagctt ctaataaaaa atatatagta 30 21 24 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 21 ctagtggggggggggggggg ggga 24 22 24 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 22 agcttccccc cccccccccc ccca 24 2328 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 23 tgaccatggc aagtcgttta gtcgtagt 28 24 15 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide24 agcaacgctt catag 15 25 16 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 25 gcgttcctgg attgtc 16 26 37 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide26 gggtctagag attcacgcga gctttggaaa ggtagca 37 27 27 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 27gtcgccatgg tgacagaacc gttaacc 27 28 19 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 28 gttcgcccgataaagggag 19 29 18 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide 29 tagcgcaacg tattactc 18 30 35 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 30gcctctagac tcatcattag atactacgac taaac 35 31 67 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 31 ctagtgggagaccacaacgg tttccctcta gaaataattt tgtttaagtt taagaagggg 60 agagaat 67 3267 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 32 catgattctc tccccttctt aaacttaaac aaaattatttctagagggaa accgttgtgg 60 tctccca 67 33 31 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 33 gcggaattcatacttattta tcattagaaa g 31 34 30 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 34 gcgccatggt aaatgaaaga aagaactaaa30 35 30 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 35 gcgtctagat caaccgaaat tcaattaagg 30 36 27 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide36 cgcaagcttc aatggaagca atgataa 27 37 28 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 37 catggcttcctcagttcttt cctctgca 28 38 20 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 38 gaggaaagaa ctgaggaagc 20

1. A plant comprising in its nuclear genome at least one heterologousexpression cassette or parts thereof comprising a nucleotide sequenceencoding a trehalose biosynthetic enzyme under control of an induciblepromoter.
 2. A plant according to claim 1, wherein said plant hasinducible drought-resistance.
 3. A plant according to claim 1, whereinsaid heterologous expression cassette comprises a nucleotide sequenceencoding a trehalose 6-phosphate synthase.
 4. A plant according to claim1, wherein said heterologous expression cassette comprises a nucleotidesequence encoding a trehalose 6-phosphate phosphatase.
 5. A plantaccording to claim 1, wherein said nucleotide sequence encoding atrehalose biosynthetic enzyme is derived from a plant, a yeast or abacteria.
 6. A plant according to claim 1, wherein said nucleotidesequence encoding a trehalose biosynthetic enzyme comprises the E. coliOtsA gene or OtsB gene.
 7. A plant according to claim 1 comprising afirst heterologous expression cassette or parts thereof comprising anucleotide sequence encoding a trehalose 6-phosphate synthase undercontrol of an inducible promoter and a second heterologous expressioncassette or parts thereof comprising a nucleotide sequence encoding atrehalose-6-phosphate phosphatase under control of an induciblepromoter.
 8. A plant according to claim 1, wherein said promoter is achemically or wound inducible promoter.
 9. A plant according to claim 8,wherein said promoter is the tobacco PR-1a promoter or the ArabidopsisPR-1 promoter.
 10. Seeds of a plant according to claim 1 or of theprogeny thereof.
 11. A plant nuclear expression cassette comprising anucleotide sequence encoding a trehalose biosynthetic enzyme undercontrol of an inducible promoter capable of directing the expression ofsaid nucleotide sequence in a plant.
 12. A recombinant vector comprisinga nucleotide sequence encoding a trehalose biosynthetic enzyme undercontrol of an inducible promoter capable of directing the expression ofsaid nucleotide sequence in a plant.
 13. A plant comprising in itsplastid genome at least one heterologous expression cassette or partsthereof comprising a nucleotide sequence encoding a trehalosebiosynthetic enzyme under control of a promoter capable of directing theexpression of said nucleotide sequence in a plastid of said plant.
 14. Aplant according to claim 13, wherein said heterologous expressioncassette comprises a nucleotide sequence encoding a trehalose6-phosphate synthase.
 15. A plant according to claim 13, wherein saidheterologous expression cassette comprises a nucleotide sequenceencoding a trehalose 6-phosphate phosphatase.
 16. A plant according toclaim 13, wherein said nucleotide sequence encoding a trehalosebiosynthetic enzyme is derived from a plant, a yeast or a bacteria. 17.A plant according to claim 13, wherein said nucleotide sequence encodinga trehalose biosynthetic enzyme comprises the E. coli OtsA gene or OtsBgene.
 18. A plant according to claim 13, wherein said promoter comprisesa transactivator-regulated promoter.
 19. A plant according to claim 18further comprising a heterologous nuclear expression cassette or partsthereof comprising a promoter operably linked to a nucleotide sequenceencoding said transactivator, wherein said promoter is capable ofdirecting the expression of said transactivator in said plant, whereinsaid transactivator is fused to a plastid targeting sequence.
 20. Aplant according to claim 19, wherein said transactivator-regulatedpromoter comprises a T7 gene 10 promoter and said transactivatorcomprises a T7 RNA polymerase.
 21. A plant according to claim 19,wherein said promoter capable of directing the expression of saidtransactivator in said plant is an inducible promoter, a tissue-specificpromoter or a constitutive promoter.
 22. A plant according to claim 21,wherein said inducible promoter is chemically or wound inducible.
 23. Aplant according to claim 22, wherein said promoter is the tobacco PR-1apromoter or the Arabidopsis PR-1 promoter.
 24. A plant according toclaim 13, wherein said promoter is transcribed by a RNA polymerasenormally present in a plastid of said plant.
 25. A plant according toclaim 24, wherein said RNA polymerase is nuclear-encoded polymerase or aplastid-encoded polymerase.
 26. A plant according to claim 25, whereinsaid promoter is a cIpP promoter, a 16S r-RNA gene promoter, a psbApromoter or a rbcL promoter.
 27. A plant according to claim 13comprising a first expression cassette comprising a nucleotide sequenceencoding a trehalose 6-phosphate synthase under control of a promotercapable of directing the expression of said nucleotide sequence in aplastid of said plant and second expression cassette comprising anucleotide sequence encoding a trehalose-6-phosphate phosphatase undercontrol of a promoter capable of directing the expression of saidnucleotide sequence in a plastid of said plant.
 28. A plant according toclaim 13, wherein said expression cassette comprises a first nucleotidesequence encoding a trehalose 6-phosphate synthase and a secondnucleotide sequence trehalose-6-phosphate phosphatase.
 29. A plantaccording to claim 28, wherein said first and second nucleotidesequences are transcribed from a single promoter in an operon-likepolycistronic gene, wherein said promoter is capable of directing theexpression of said operon-like polycistronic gene in a plastid of saidplant.
 30. Seeds of a plant according to claim 13 or of the progenythereof.
 31. A plastid expression cassette comprising a nucleotidesequence encoding a trehalose biosynthetic enzyme under control of apromoter capable of directing the expression of said nucleotide sequencein a plastid of a plant.
 32. A recombinant vector comprising anucleotide sequence encoding a trehalose biosynthetic enzyme undercontrol of a promoter capable of directing the expression of saidnucleotide sequence in a plastid of a plant.
 33. A plant comprising inits plastid genome two or more genes transcribed from a single promoterin an operon-like polycistronic gene, wherein said promoter is capableof directing the expression of said operon-like polycistronic gene in aplastid of said plant, wherein said operon-like polycistronic genefurther comprises a heterologous intervening DNA sequence between twogenes in said operon-like polycistronic gene.
 34. A plant according toclaim 33, wherein said intervening DNA sequence comprises a portion of anon-eukaryotic 5′UTR.
 35. A plant according to claim 34, wherein said5′UTR is derived from a virus.
 36. A plant according to claim 32,wherein said intervening DNA is modified to prevent the formation of RNAsecondary structures in a transcript of said operon-like polycistronicgene.
 37. A plant according to claim 32, wherein said operon-likepolycistronic gene comprises a gene comprising a nucleotide sequenceencoding at least one trehalose biosynthetic enzyme.
 38. Seeds of aplant according to claim 32 or of the progeny thereof.
 39. A method ofproducing a plant according to claim 18 comprising: pollinating a plantcomprising a heterologous plastid expression cassette or parts thereofcomprising a transactivator-mediated promoter operably linked to anucleotide sequence encoding at least one trehalose biosynthetic enzymewith pollen from a plant comprising a heterologous nuclear expressioncassette or parts thereof comprising a promoter operably linked to anucleotide sequence encoding a transactivator capable of regulating saidtransactivator-mediated promoter, wherein said promoter operably linkedto a DNA sequence coding for a transactivator is capable of directingthe expression of said transactivator in said plant, wherein saidtransactivator is fused to a plastid targeting sequence; recovering seedfrom the plant thus pollinated; and cultivating a plant as describedabove from said seed.
 40. A method for producing trehalose in a plant byexpressing at least one heterologous nucleotide sequence encoding atrehalose biosynthetic enzyme under the control of an inducible promoterin the nuclear genome of said plant or by expressing at least oneheterologous nucleotide sequence encoding a trehalose biosyntheticenzyme in a plastid of said plant.
 41. A method comprising expressing atleast one heterologous nucleotide sequence encoding a trehalosebiosynthetic enzyme under the control of an inducible promoter in thenuclear genome of a plant or expressing at least one heterologousnucleotide sequence encoding a trehalose biosynthetic enzyme in aplastid of a plant, wherein the expression of said nucleotide sequencein said plant confers upon said plant a trait selected from the groupconsisting of drought-resistant, increased storage properties of theharvested plant, improved shelf-life of fruits, vegetables or flowersderived from said plant or stabilization of proteins expressed in saidplant.
 42. A method of expressing two or more genes from a singlepromoter in a plastid of a plant comprising introducing into the plastidgenome of said plant a operon-like polycistronic gene comprising saidtwo or more genes operably linked to a promoter capable of expressingsaid operon-like polycistronic gene in a plastid of said plant, whereinsaid operon-like polycistronic gene further comprises an intervening DNAsequence between two genes.